88kDa tumorigenic growth factor and antagonists

ABSTRACT

This invention relates to products and methods for treating cancer and for diagnosing tumorigenicity and other diseases associated with alteration in GP88 expression or action. Antagonists to an 88 KDa autocrine growth and tumorigenicity stimulator are provided which inhibit its expression or biological activity. The antagonists include antisense oligonucleotides and antibodies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of Ser. No. 09/813,156 filed on Mar.21, 2001 now U.S. Pat. No. 6,670,183 which is a continuation of Ser. No.08/991,862 filed on Dec. 16, 1997, now U.S. Pat. No. 6,309,826 issued onOct. 30, 2001, which is a continuation-in-part of Ser. No. 08/863,079filed on May 23, 1997 now abandoned.

FIELD OF THE INVENTION

This invention relates to cell biology, physiology and medicine, andconcerns an 88 kDa glycoprotein growth factor (“GP88”) and compositionsand methods which affect the expression and biological activity of GP88.These compositions and methods are useful for diagnosis and treatment ofdiseases including cancer.

REFERENCES

Several publications are referenced herein by Arabic numerals withinparenthesis. Full citations for these references may be found at the endof the specification immediately preceding the claims.

BACKGROUND OF THE INVENTION

The proliferation and differentiation of cells in multicellularorganisms is subject to a highly regulated process (1). A distinguishingfeature of cancer cells is the absence of control over this process;proliferation and differentiation become deregulated resulting inuncontrolled growth. Significant research efforts have been directedtoward better understanding this difference between normal and tumorcells. One area of research focus is growth factors and, morespecifically, autocrine growth stimulation.

Growth factors are polypeptides which carry messages to cells concerninggrowth, differentiation, migration and gene expression (2). Typically,growth factors are produced in one cell and act on another cell tostimulate proliferation. However, certain malignant cells, in culture,demonstrate a greater or absolute reliance on an autocrine growthmechanism (3). Malignant cells which observe this autocrine behaviorcircumvent the regulation of growth factor production by other cells andare therefore unregulated in their growth.

Study of autocrine growth control advances understanding of cell growthmechanisms and leads to important advances in the diagnosis andtreatment of cancer. Toward this end, a number of growth factors havebeen studied, including insulin-like growth factors (“IGF1” and “IGF2”),gastrin-releasing peptide (“GRP”), transforming growth factors alpha andbeta (“TGF-a” and “TGF-b”), and epidermal growth factor (“EGF”).

The present invention is directed to a recently discovered growthfactor. This growth factor was first discovered in the culture medium ofhighly tumorigenic “PC cells,” an insulin-independent variant isolatedfrom the teratoma derived adipogenic cell line 1246. This growth factoris referred to herein as “GP88.” GP88 has been purified and structurallycharacterized (4). Amino acid sequencing of GP88 indicates that GP88 hasamino acid sequence similarities with the mouse granulin/epithelinprecursor.

Granulins/epithelins (“grn/epi”) are 6 kDa polypeptides and belong to anovel family of double cysteine rich polypeptides (5, 6). U.S. Pat. No.5,416,192 (Shoyab et al.) is directed to 6 kDa epithelins, particularlyepithelin 1 and epithelin 2. According to Shoyab, both epithelins areencoded by a common 63.5 kDa precursor, which is processed into smallerforms as soon as it is synthesized, so that the only natural productsfound in biological samples are the 6 kDa forms. Shoyab et al. teachesthat the epithelin precursor is biologically inactive.

Contrary to the teachings of Shoyab et al., the inventor's laboratoryhas demonstrated that the precursor is not always processed as soon asit is synthesized. Studies, conducted in part by this inventor, havedemonstrated that the precursor (i.e., GP88) is in fact secreted as an88 kDa glycoprotein with an N-linked carbohydrate moiety of 20 kDa (4).Analysis of the N-terminal sequence of GP88 indicates that GP88 startsat amino acid 17 of the grn/epi precursor, demonstrating that the first17 amino acids from the protein sequence deduced from the precursor cDNAcorrespond to a signal peptide compatible with targeting for membranelocalization or for secretion (4).

Also in contrast to the teachings of Shoyab et al., GP88 is biologicallyactive and has growth promoting activity, particularly as an autocrinegrowth factor for the producer cells.

SUMMARY OF INVENTION

The inventor has now unexpectedly discovered that a glycoprotein (GP88),which is expressed in a tightly regulated fashion in normal cells, isoverexpressed and unregulated in highly tumorigenic cells derived fromthe normal cells, that GP88 acts as a stringently required growthstimulator for the tumorigenic cells and that inhibition of GP88expression or action in the tumorigenic cells results in an inhibitionof the tumorigenic properties of the overproducing cells.

It is an object of this invention to provide GP88 antagonizingcompositions capable of inhibiting the expression or activity of GP88.

A further object of the invention is to provide methods for treatingdiseases associated with a defect in GP88 quantity or activity such asbut not limited to cancer in mice or humans.

Another object of the invention is to provide methods for determiningthe susceptibility of a subject to diseases associated with a defect inGP88 expression or action.

Yet another object of the invention is to provide methods for measuringsusceptibility to GP88 antagonizing therapy.

Yet another object of the invention is to provide methods, reagents, andkits for the in vitro and in vivo detection of GP88 and tumorigenicactivity in cells.

Additional objects and advantages of the invention will be set forth inpart in the description that follows, and in part will be obvious fromthe description, or may be learned by the practice of the invention.

To achieve the objects and in accordance with the purpose of theinvention, as embodied and properly described herein, the presentinvention provides compositions for diagnosis and treatment of diseasessuch as but not limited to cancer in which cells exhibit an alteredexpression of GP88 or altered response to GP88.

Use of the term “altered expression” herein means increased expressionor overexpression of GP88 by a factor of at least two-fold, and at timesby a factor of 10 or more, based on the level of mRNA or protein ascompared to corresponding normal cells or surrounding peripheral cells.The term “altered expression” also means expression which becameunregulated or constitutive without being necessarily elevated. Use ofthe terms increased or altered “response” to GP88 means a conditionwherein increase in any of the biological functions (e.g., growth,differentiation, viral infectivity) conferred by GP88 results in thesame or equivalent condition as altered expression of GP88.

Use of the term “GP88” herein means epithelin/granulin precursor in cellextracts and extracellular fluids, and is intended to include not onlyGP88 according to the amino acid sequences included in FIG. 8 or 9,which are of mouse and human origins, but also GP88 of other species. Inaddition, the term also includes functional derivatives thereof havingadditional components such as a carbohydrate moiety including aglycoprotein or other modified structures.

Also intended by the term GP88 is any polypeptide fragment having atleast 10 amino-acids present in the above mentioned sequences. Sequencesof this length are useful as antigens and for making immunogenicconjugates with carriers for the production of antibodies specific forvarious epitopes of the entire protein. Such polypeptides are useful inscreening such antibodies and in the methods directed to detection ofGP88 in biological fluids. It is well known in the art that peptides areuseful in generation of antibodies to larger proteins (7). In oneembodiment of this invention, it is shown that peptides from 12-19amino-acids in length have been successfully used to develop antibodiesthat recognize the full length GP88.

The polypeptide of this invention may exist covalently or non-covalentlybound to another molecule. For example, it may be fused to one or moreother polypeptides via one or more peptide bonds such as glutathionetransferase, poly-histidine, or myc tag.

The polypeptide is sufficiently large to comprise an antigeneticallydistinct determinant or epitope which can be used as an immunogen toreproduce or test antibodies against GP88 or a functional derivativethereof.

One embodiment includes the polypeptide substantially free of othermammalian peptides. GP88 of the present invention can be biochemicallyor immunochemically purified from cells, tissues or a biological fluid.Alternatively, the polypeptide can be produced by recombinant means in aprokaryotic or eukaryotic expression system and host cells.

“Substantially free of other mammalian polypeptides” reflects the factthat the polypeptide can be synthesized in a prokaryotic or anon-mammalian or mammalian eukaryotic organism, if desired.Alternatively, methods are well known for the synthesis of polypeptidesof desired sequences by chemical synthesis on solid phase supports andtheir subsequent separation from the support. Alternatively, the proteincan be purified from tissues or fluids of mammals where it naturallyoccurs so that it is at least 90% pure (on a weight basis) or even 99%pure, if desired, of other mammalian polypeptides, and is thereforesubstantially free from them. This can be achieved by subjecting thetissue extracts or fluids to standard protein purification such as onimmunoabsorbants bearing antibodies reactive against the protein. Oneembodiment of the present invention describes purification methods forthe purification of naturally occurring GP88 and of recombinant GP88expressed in baculovirus infected insect cells. Alternatively,purification from such tissues or fluids can be achieved by acombination of standard methods such as but not limited to the onesdescribed in reference (4).

As an alternative to a native purified or recombinant glycoprotein orpolypeptide, “GP88” is intended to also include functional derivatives.By functional derivative is meant a “fragment,” “variant,” “analog,” or“chemical derivative” of the protein or glycoprotein as defined below. Afunctional derivative retains at least a portion of the function of thefull length GP88 which permits its utility in accordance with thepresent invention.

A “fragment” of GP88 refers to any subset of the molecule that is ashorter peptide. This corresponds for example but is not limited toregions such as K19T and S14R for mouse GP88, and E19V and A14R(equivalent to murine K19T and S14R, respectively) for human GP88.

A “variant” of GP88 refers to a molecule substantially similar to eitherthe entire peptide or a fragment thereof. Variant peptides may beprepared by direct chemical synthesis of the variant peptide usingmethods known in the art.

Alternatively, amino acid sequence variants of the peptide can beprepared by modifying the DNA which encodes the synthesized protein orpeptide. Such variants include, for example, deletions, insertions, orsubstitutions of residues within the amino-acid sequence of GP88. Anycombination of deletion, insertion, and substitution may also be made toarrive at the final construct, provided the final construct possessesthe desired activity. The mutation that will be made in the DNA encodingthe variant peptide must not alter the reading frame and preferably willnot create complementary regions that could produce secondary mRNAstructures. At the genetic level these variants are prepared by sitedirected mutagenesis (8) of nucleotides in the DNA encoding the peptidemolecule thereby producing DNA encoding the variant, and thereafterexpressing the DNA in recombinant cell culture. The variant typicallyexhibits the same qualitative biological activity as the nonvariantpeptide.

An “analog” of GP88 protein refers to a non-natural moleculesubstantially similar to either the entire molecule or a fragmentthereof.

A “chemical derivative” contains additional chemical moieties notnormally a part of the peptide or protein. Covalent modifications of thepeptide are also included within the scope of this invention. Suchmodifications may be introduced into the molecule by reacting targetedamino-acid residues of the peptide with an organic derivatizing agentthat is capable of reacting with selected side chains or terminalamino-acid residues. Most commonly derivatized residues are cysteinyl,histidyl, lysinyl, arginyl, tyrosyl, glutaminyl, asparaginyl and aminoterminal residues. Hydroxylation of proline and lysine, phosphorylationof hydroxyl groups of seryl and threonyl residues, methylation of thealpha-amino groups of lysine, histidine, and histidine side chains,acetylation of the N-terminal amine and amidation of the C-terminalcarboxylic groups. Such derivatized moieties may improve the solubility,absorption, biological half life and the like. The moieties may alsoeliminate or attenuate any undesirable side effect of the protein andthe like. In addition, derivatization with bifunctional agents is usefulfor cross-lining the peptide to water insoluble support matrices or toother macromolecular carriers. Commonly used cross-linking agentsinclude glutaraldehyde, N-hydroxysuccinimide esters, homobifunctionalimidoesters, 1,1-bis(-diazoloacetyl)-2-phenylethane, and bifunctionalmaleimides. Derivatizing agents such asmethyl-3-[9p-azidophenyl)]dithiopropioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287 and 3,691,016 may be employed for proteinimmobilization.

Use of the term GP88 “antagonizing agents” herein means any compositionthat inhibits or blocks GP88 expression, production or secretion, or anycomposition that inhibits or blocks the biological activity of GP88.This can be achieved by any mode of action such as but not limited tothe following:

(A) GP88 antagonizing agents include any reagent or molecule inhibitingGP88 expression or production including but not limited to:

(1) antisense GP88 DNA or RNA molecules that inhibit GP88 expression byinhibiting GP88 translation;

(2) reagents (hormones, growth factors, small molecules) that inhibitGP88 mRNA and/or protein expression at the transcriptional,translational or post-transaltional levels;

(3) factors, reagents or hormones that inhibit GP88 secretion;

(B) GP88 antagonizing agents also include any reagent or molecule thatwill inhibit GP88 action or biological activity such as but not limitedto:

(1) neutralizing antibodies to GP88 that bind the protein and prevent itfrom exerting its biological activity;

(2) antibodies to the GP88 receptor that prevent GP88 from binding toits receptor and from exerting its biological activity;

(3) competitive inhibitors of GP88 binding to its receptors;

(4) inhibitors of GP88 signaling pathways.

Specific examples presented herein provide a description of preferredembodiments, particularly the use of neutralizing antibodies to inhibitGP88 biological action and the growth of the highly tumorigenic PCcells; the use of antisense GP88 cDNA and antisense GP88oligonucleotides to inhibit GP88 expression leading to inhibition of thetumorigenic properties of the PC cells; characterization of GP88receptors on cell surfaces of several cell lines including the mammaryepithelial cell line C57MG, the 1246 and PC cell lines and the mink lungepithelial cell line CCL64.

In one embodiment of the invention, the GP88 antagonizing agents areantisense oligonucleotides to GP88. The antisense oligonucleotidespreferably inhibit GP88 expression by inhibiting translation of the GP88protein.

Alternatively, such a composition may comprise reagents, factors orhormones that inhibit GP88 expression by regulating GP88 genetranscriptional activity. Such a composition may comprise reagents,factors or hormones that inhibit GP88 post-translational modificationand its secretion. Such a composition may comprise reagents that act asGP88 antagonists that block GP88 activity by competing with GP88 forbinding to GP88 cell surface receptors. Alternatively, such acomposition may comprise factors or reagents that inhibit the signalingpathway transduced by GP88 once binding to its receptors on diseasedcells.

Alternatively, the composition may comprise reagents that block GP88action such as an antibody specific to GP88 that neutralizes itsbiological activity, or an antibody to the GP88 receptor that blocks itsactivity.

The antibodies of the invention (neutralizing and others) are preferablyused as a treatment for cancer or other diseases in cells which exhibitan increased expression of GP88. By the term “neutralizing” it shall beunderstood that the antibody has the ability to inhibit or block thenormal biological activity of GP88, including GP88's ability tostimulate cell proliferation or to induce tumor growth in experimentalanimals and in humans. An effective amount of anti-GP88 antibody isadministered to an animal, including humans, by various routes. In analternative embodiment, the anti-GP88 antibody is used as a diagnosticto detect cells which exhibit an altered (increased) expression of GP88as occurring in diseases such as but not limited to cancers, and toidentify diseased cells whose growth is dependent on GP88 and which willrespond to GP88 antagonizing therapy. In yet another embodiment, theanti-GP88 antibody is used to deliver compounds such as cytotoxicfactors or antisense oligonucleotides to cells expressing or responsiveto GP88.

The antisense oligonucleotides of the invention are also used as atreatment for cancer in cells which exhibit an increased expression ofGP88. An effective amount of the antisense oligonucleotide isadministered to an animal, including humans, by various routes.

The present invention also provides a method for determining thesusceptibility to diseases associated with a defect in GP88 expressionor action which comprises obtaining a sample of biological fluid ortissue and measuring the amount of GP88 in the fluid or tissue ormeasuring the susceptibility of the cells to respond to GP88. In thecase of cancer, the amount of GP88 being proportional to thesusceptibility to the cancer.

The present invention also provides a method for measuring the degree ofseverity of cancer which comprises obtaining a sample of biologicalfluid or tissue and measuring the amount of GP88 in the fluid or tissuesample, the amount of GP88 being proportional to the degree or severityof the cancer.

The present invention also provides a method for measuringsusceptibility to GP88 antagonizing therapy which comprises obtaining asample of the diseased tissue (biopsy), maintaining the cells derivedfrom the sample in culture, treating the cells derived from the culturewith anti-GP88 neutralizing antibody and determining if the neutralizingantibody inhibits the cell growth. Ability of the antibody to inhibitcell growth is indicative that the cells are dependent on GP88 toproliferate and is predictive that GP88 antagonizing therapy will beefficacious.

The present invention also provides a method for determining thesusceptibility to cancer associated with an abnormality in GP88 receptorlevel or activity which comprises obtaining a sample of tissue andmeasuring the amount of GP88 receptor protein or mRNA in the tissue ormeasuring the tyrosine kinase activity of the receptor in the tissue(GP88 binding to its receptor induces tyrosine phosphorylation ofcellular proteins including the receptor for G88).

The present invention also provides a method for targeting GP88antagonizing reagents to the diseased site by conjugating them to ananti-GP88 antibody or an anti-GP88 receptor antibody.

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate embodiments of the invention and,together with the description, serve to explain the principles of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A compares the level of expression of GP88 protein in the 1246,1246-3A and PC cell lines. Cells were cultured in DME-F12 mediumsupplemented with 2% fetal bovine serum (FBS). GP88 expression levelswere measured by immunoprecipitation and Western blot analysis withanti-K19T antibody.

FIG. 1B compares the level of GP88 mRNA expression in the 1246, 1246-3Aand PC cell lines. mRNA for RPL32 is used as an internal control forequal amounts of RNA loading.

FIG. 1C compares the expression of GP88 mRNA in 1246 cells (left panel)and in PC cells (right panel) in serum-free and serum containing medium.The results show that GP88 expression in 1246 cells is inhibited by theaddition of fetal bovine serum whereas such inhibition is not observedin the highly tumorigenic PC cells.

FIG. 2 illustrates the effect of treatment of the highly tumorigenic PCcells with increasing concentrations of anti-GP88 neutralizing antibody.

FIG. 3 shows C3H mice injected subcutaneously with 106 antisense GP88transfected PC cells (bottom) and with empty vector transfected controlPC cells (top).

FIG. 4 shows in vivo GP88 expression levels in C3H mice tumor tissuesand in surrounding normal tissues.

FIG. 5 shows GP88 mRNA expression levels in estrogen receptor positiveand estrogen receptor negative human mammary carcinoma cell lines.

FIG. 6 shows the effect of increasing concentrations of GP88 on thegrowth of the mouse mammary epithelial cell line C57.

FIG. 7 shows the growth properties and tumorigenic ability of PC cellstransfected with a cytomegalovirus promoter controlled expression vectorcontaining GP88 in antisense orientation and PC cells transfected withan empty vector.

FIG. 8 shows the nucleotide and deduced amino-acid sequence of mouseGP88 (SEQ ID NOS 1 & 2, respectively). Peptide regions used as antigensto raise anti-GP88 antibodies K19T and S14R are underlined. The regioncloned in the antisense orientation in the pCMV4 mammalian expressionvector is indicated between brackets.

FIG. 9A shows the nucleotide sequence of human GP88 cDNA (SEQ ID NOS 16& 17, respectively). Indicated between brackets is the region cloned inthe antisense orientation into the pcDNA3 mammalian expression system;and

FIG. 9B shows the deduced amino-acid sequence of human GP88. The E19Vregion used as antigen to develop anti-human GP88 neutralizing antibodyis underlined. It also indicates the region A14R equivalent to the mouseS14R region.

FIG. 10 shows the amino-acid sequence of mouse GP88 (SEQ ID NO: 2)arranged to show the 7 and one-half repeats defined as granulins g, f,B, A, C, D and e (right side). This representation shows that the regionK19T and S14R used to raise GP88 antibodies for developing anti-GP88neutralizing antibodies is found between two epithlin/granulin repeatsin what is considered a variant region. Indicated on the right hand sideis the granulin classification of the repeats according to Bateman et al(6). Granulin B and granulin A are also defined as epithelin 2 andepithelin 1 respectively according to Plowman et al., 1992 (5).

FIG. 11 shows a schematic representation of pCMV4 and a GP88 cDNA cloneindicating the restriction sites used to clone GP88 antisense cDNA intothe expression vector.

FIG. 12 shows the cross-linking of ¹²⁵I-rGP88 to GP88 cell surfacereceptors on CCL-64 cells. The cross-linking reaction was carried outwith disuccinimidyl suberate (DSS). Reaction products were analyzed bySDS-PAGE on a 7% polyacrylamide gel.

FIG. 13 shows the cross-linking of ¹²⁵I-rGP88 to GP88 cell surfacereceptors on 3T3 fibroblasts, PC cells and C57MG mammary epithelialcells. The results show that these various cell lines display GP88 cellsurface receptors of similar molecular weight as the ones on CCL64 cells(FIG. 12).

FIG. 14 shows GP88 expression levels in non-tumorigenic MCF 10A and inmalignant (MCF 7, MDA-468) human mammary epithelial cells.

FIG. 15 shows that GP88 expression is inhibited by antisense GP88 cDNAtransfection in human breast carcinoma MDA-468 cells.

DESCRIPTION OF THE INVENTION

Reference will now be made in detail to the presently preferredembodiments of the invention, which, together with the followingexamples, serve to explain the principles of the invention.

Biological Activity of GP88

The invention relates to GP88 and antitumor and antiviral compositionsuseful for treating and diagnosing diseases linked to altered(increased) expression of GP88. Alternatively this invention is used fortreating and diagnosing diseases linked to increased responsiveness toGP88. Using a murine model system consisting of three cell lines, theinventor has shown that cells which overexpress GP88 form tumors. Theparent cell line, 1246, is a C3H mouse adipogenic cell line whichproliferates and differentiates into adipocytes in a defined mediumunder stringent regulation by insulin (9, 10). The 1246 cells cannotform tumors in a syngeneic animal (C3H mouse) even when injected at ahigh cell density. An insulin independent cell line, 1246-3A, wasisolated from 1246 cells maintained in insulin-free medium (11). The1246-3A cells lost the ability to differentiate and form tumors when 10⁶are injected subcutaneously in syngeneic mice. A highly tumorigenic cellline, PC, was developed from 1246-3A cells by an in vitro-in vivoshuttle technique. The PC cells formed tumors when 10⁴ cells wereinjected into syngeneic mice (12).

GP88 is overexpressed in the insulin-independent tumorigenic cell linesrelative to the parent non-tumorigenic insulin-dependent cell line.Moreover, the degree of overexpression of GP88 positively correlateswith the degree of tumorigenicity of these cells, demonstrating for thefirst time that GP88 is important in tumorigenesis (FIG. 1). Withreference to FIG. 1, since GP88 is synthesized by cells but alsosecreted in culture medium, the level of GP88 was determined in celllysates and in culture medium (CM). All cells were cultivated in DME/F12nutrient medium supplemented with 2% fetal bovine serum. When cellsreached confluency, culture medium (CM) was collected and cell lysateswere prepared by incubation in buffer containing detergent followed by a10,000×g centrifugation. Cell lysate and conditioned medium werenormalized by cell number. Samples from cell lysate and conditionedmedium were analyzed by Western blot analysis using an anti-GP88antibody, as explained below.

The development of a neutralizing antibody confirmed GP88's key role intumorigenesis. When an anti-GP88 antibody directed to the K19T region ofmouse GP88 was added to the culture medium, the growth of highlytumorigenic PC cells was inhibited in a dose dependent fashion (FIG. 2).With reference to FIG. 2, PC cells were cultivated in 96 well plates ata density 2×10⁴ cells/well in DME/F12 medium supplemented with humanfibronectin (2 μg/ml) and human transferrin (10 μg/ml). Increasingconcentrations of anti-GP88 IgG fraction were added to the wells afterthe cells were attached. Control cells were treated with equivalentconcentrations of non-immune IgG. Two days later, 0.25 mCi of³H-thymidine was added per well for 6 hrs. Cells were then harvested tocount ³H-thymidine incorporated into DNA as a measure for cellproliferation.

Moreover, when the expression of GP88 was specifically inhibited byantisense GP88 cDNA in PC cells, the production of GP88 was reduced andthese PC cells could no longer form tumors in syngeneic C3H mouse. Inaddition, these PC cells regained responsiveness to insulin. Withreference to FIG. 3 and Tables 1 and 2, C3H female mice were injectedsubcutaneously with 10⁶ antisense GP88 transfected PC cells (asexplained below) or 10⁶ empty vector transfected PC cells. Mice weremonitored daily for tumor appearance. Photographs were taken 45 daysafter injection of the cells. The results show that mice injected withantisense GP88 PC cells do not develop tumors, in contrast to the miceinjected with empty vector transfected PC cells used as control.

TABLE 1 COMPARISON OF TUMORIGENIC PROPERTIES OF GP88 ANTISENSETRANSFECTED CELLS, CONTROL TRANSFECTED CELLS AND PC CELLS AVERAGE DAY OFNUMBER OF AVERAGE CELLS TUMOR MICE WITH TUMOR INJECTED DETECTION TUMORSWEIGHT (g) PC 15 ± 3.0 5/5 9.0 ± 3.2 P14 15 ± 3.7 5/5 7.8 ± 2.7 ASGP88 —0/5 — PC: Control non-transfected cells P-14: Empty vector controltransfected PC cells ASGP88: PC cells transfected with expression vectorcontaining GP88 antisense cDNA

Tumors were excised and weighed at 45 days. —indicates no tumorformation.

TABLE 2 COMPARISON OF PROPERTIES OF 1246, PC CELLS AND GP88 ANTISENSECELLS insulin GP88 antisense independence transfection 1246 cells PCcells Antisense GP 88 cells insulin insulin-independent for recovery ofinsulin responsive for growth differentiation responsiveness for growthgrowth and deficient (differentiation?) differentiation autocrineproduction of insulin-related factor cell surface cell surface insulinreceptor cell surface insulin receptor insulin receptor expression verylow expression elevated expression high GP88 GP88 expression GP88expression inhibited expression low constitutively high by antisenseGP88 No inhibition by serum expression inhibited by serum GP88 GP88expression recovery of insulin expression constitutive regulation forendogenous regulated GP88 expression by insulin non- highly tumorigenicnon-tumorigenic tumorigenic

Comparison of the expression of GP88 indicates that in vivo GP88 levelsin tumors is dramatically higher than in normal tissues (FIG. 4). C3Hmice were injected with 10⁶ PC cells. Tumor bearing mice wereeuthanized. Tumors, fat pads and connective tissue were collected. Celllysates were prepared by incubation in buffer containing detergent asdescribed above for FIG. 1. Protein concentration of tissue extracts wasdetermined, and equivalent amounts of proteins for each sample wereanalyzed by SDS-PAGE followed by Western blot analysis using anti-GP88antibody to measure the content of GP88 in tissue extracts. The resultsshowed that the level of GP88 in tumor extracts is at least 10-foldhigher than in surrounding connective and fat tissues.

In normal cells (1246 cells, fibroblasts), the expression of GP88 isregulated, in particular by insulin, and inhibited by fetal bovineserum. In tumorigenic cells, a loss of regulation of normal growth leadsto the increased expression of GP88 and the acquisition of GP88dependence for growth. Therefore, inhibition of GP88 expression and/oraction is an effective approach to suppression of tumorigenesis.Detection of an elevated GP88 expression in biopsies provides diagnosticanalysis of tumors that are responsive to GP88 inhibition therapy.

GP88 is also a tumor inducing factor in human cancers. As seen in the1246-3A cell line, a loss of responsiveness to insulin (or to IGF-I) anda concurrent increase in malignancy has been well documented (13, 14) inseveral human cancers including but not limited to breast cancers.Specifically, breast carcinoma is accompanied by the acquisition of aninsulin/IGF-I autocrine loop, which is also the starting point of thedevelopment of tumorigenic properties in the mouse model systemdiscussed above. Furthermore, GP88 expression is elevated in humanbreast carcinomas. More specifically, with reference to FIG. 5, humanGP88 was highly expressed in estrogen receptor positive and also inestrogen receptor negative insulin/IGF-I independent highly malignantcells. Also, GP88 is a potent growth factor for mammary epithelial cells(FIG. 6 ). The data in FIG. 5 was obtained by cultivating MCF7,MDA-MB-453 and MDA-MB-468 cells in DME/F12 medium supplemented with 10%fetal bovine serum (FBS). RNA was extracted from each cell line by theRNAZOL method and poly-A⁺RNA prepared. GP88 mRNA expression was examinedby Northern blot analysis with 3 μg of poly-A⁺RNA for each cell lineusing ³² P-labeled GP88 cDNA probe.

For Northern blot analysis of GP88 mRNA expression in rodent cells ortissues (mouse and rats), we used a mouse GP88 cDNA probe 311 bp inlength starting at nucleotide 551 to 862 (corresponding to amino-acidsequence 160 to 270). RNA can be extracted by a variety of methods(Sambrook, Molecular Biology manual: 35) well known to people ofordinary skill in the art. The method of choice was to extract RNA usingRNAZOL (Cinnabiotech) or TRIZOL (Gibco-BRL) solutions which consists ofa single step extraction by guanidinium isothiocyanate andphenol-chloroform.

For Northern blot analysis of GP88 mRNA expression in human cell lines,a 672 bp human GP88 cDNA probe was developed corresponding to nucleotide1002 to 1674 (corresponding to amino-acid sequence 334-558) of humanGP88. See example 8 for a detailed and specific description of theNorthern blot analysis method used in the preferred embodiments.

With respect to FIG. 6, C57MG cells were cultivated in the presence ofincreasing concentrations of GP88 purified from PC cells conditionedmedium (top panel), and recombinant GP88 expressed in insect cells(bottom panel), to demonstrate the growth stimulating effect ofincreasing concentrations of GP88 on the growth of the mouse mammaryepithelial cell line C57MG.

A correlation between IGF-1 autocrine production and increasedmalignancy has also been well established for glioblastomas,teratocarcinomas and breast carcinomas (2, 15, 16, 17). In thesecancers, GP88 expression is also elevated in human tumors when comparedto non-tumorigenic human fibroblasts and other human cell lines. GP88promotes the growth of mammary carcinoma cells.

Anti-GP88 Antibodies

The invention provides compositions for treating and diagnosing diseaseslinked to increased expression of GP88. This also will apply totreatment and diagnosis of diseases linked to increased responsivenessto GP88. The compositions of this invention include anti-GP88 antibodieswhich neutralize the biological activity of GP88.

The present invention is also directed to an antibody specific for anepitope of GP88 and the use of such antibody to detect the presence ormeasure the quantity or concentration of GP88 molecule, a functionalderivative thereof or a homologue from different animal species in acell, a cell or tissue extract, culture medium or biological fluid.Moreover, antibody can be used to target cytotoxic molecules to aspecific site.

For use as antigen for development of antibodies, the GP88 proteinnaturally produced or expressed in recombinant form or functionalderivative thereof, preferably having at least 9 amino-acids, isobtained and used to immunize an animal for production of polyclonal ormonoclonal antibody. An antibody is said to be capable of binding amolecule if it is capable of reacting with the molecule to thereby bindthe molecule to the antibody. The specific reaction is meant to indicatethat the antigen will react in a highly selective manner with itscorresponding antibody and not with the multitude of other antibodieswhich may be evoked by other antigens.

The term antibody herein includes but is not limited to human andnon-human polyclonal antibodies, human and non-human monoclonalantibodies (mAbs), chimeric antibodies, anti-idiotypic antibodies(anti-IdAb) and humanized antibodies. Polyclonal antibodies areheterogeneous populations of antibody molecules derived either from seraof animals immunized with an antigen or from chicken eggs. Monoclonalantibodies (“mAbs”) are substantially homogeneous populations ofantibodies to specific antigens. mAbs may be obtained by methods knownto those skilled in the art (18, 19, 20 and U.S. Pat. No. 4,376,110).Such antibodies may be of any immunological class including IgG, IgM,IgE, IgA, IgD and any subclass thereof. The hybridoma producing humanand non-human antibodies to GP88 may be cultivated in vitro or in vivo.For production of a large amount of mAbs, in vivo is the presentlypreferred method of production. Briefly, cells from the individualhybridomas are injected intraperitoneally into pristane primed Balb/cmice or Nude mice to produce ascites fluid containing highconcentrations of the desired mAbs. mAbs may be purified from suchascites fluids or from culture supernatants using standardchromatography methods well known to those of skill in the art.

Human monoclonal Ab to human GP88 can be prepared by immunizingtransgenic mice expressing human immunoglobulin genes. Hybridomaproduced by using lymphocytes from these transgenic animals will producehuman immunoglobulin instead of mouse immunoglobulin.

Since most monoclonal antibodies are derived from murine source andother non-human sources, their clinical efficiency may be limited due tothe immunogenicity of rodent mAbs administered to humans, weakrecruitment of effector function and rapid clearance from serum (25). Tocircumvent these problems, the antigen-binding properties of murineantibodies can be conferred to human antibodies through a process calledhumanization (25). A humanized antibody contains the amino-acidsequences for the 6 complementarity-determining regions (CDRs) of theparent murine mAb which are grafted onto a human antibody framework. Thelow content of non-human sequences in humanized antibodies (around 5%)has proven effective in both reducing the immunogenicity and prolongingthe serum half life in humans. Methods such as the ones using monovalentphage display and combinatorial library strategy (26, 27) forhumanization of monoclonal antibodies are now widely applied to thehumanization of a variety of antibodies and are known to people skilledin the art. These humanized antibodies and human antibodies developedwith transgenic animals as described above are of great therapeutic usefor several diseases including but not limited to cancer.

Hybridoma supernatants and sera are screened for the presence ofantibody specific for GP88 by any number of immunoassays including dotblots and standard immunoassays (EIA or ELISA) which are well known inthe art. Once a supernatant has been identified as having an antibody ofinterest, it may be further screened by Western blotting to identify thesize of the antigen to which the antibody binds. One of ordinary skillin the art will know how to prepare and screen such hybridomas withoutundue experimentation in order to obtain a desired polyclonal or mAb.

Chimeric antibodies have different portions derived from differentanimal species. For example, a chimeric antibody might have a variableregion from a murine mAb and a human immunoglobulin constant region.Chimeric antibodies and methods for their production are also known tothose skilled in the art (21-24).

An anti-idiotypic (“anti-IdAb”) is an antibody which recognizes uniquedeterminants generally associated with the antigen-binding site of anantibody. An anti-IdAb can be prepared by immunizing an animal of thesame species and genetic type (e.g., mouse strain) as the source of themAb with the mAb to which an anti-IdAb is being prepared. The immunizedanimal will recognize and respond to the idiotypic determinants of theimmunizing antibody by producing antibody to these idiotypicdeterminants (the anti-IdAb). The anti-IdAb may also be used as animmunogen to produce an immune response in yet another animal, producinga so-called anti-anti-IdAb. The anti-anti-IdAb may be epitopicallyidentical to the original mAb which induced the anti-IdAb. Thus by usingantibodies to the idiotypic determinants of a mAb, it is possible toidentify other clones expressing antibodies of identical specificity.

Accordingly, mAbs generated against GP88 may be used to induce human andnon-human anti-IdAbs in suitable animals. Spleen cells from suchimmunized mice are used to produce hybridomas secreting human ornon-human anti-Id mAbs. Further, the anti-Id mAbs can be coupled to acarrier such as Keyhole Limpet Hemocyanin (KLH) or bovine serum albumin(BSA) and used to immunize additional mice. Sera from these mice willcontain human or non-human anti-anti-IdAb that have the bindingproperties of the original mAb specific for a GP88 polypeptide epitope.The anti-Id mAbs thus have their own idiotypic epitopes or idiotypesstructurally similar to the epitope being evaluated.

The term antibody is also meant to include both intact molecules as wellas fragments thereof such as, for example, Fab and F(ab′)₂, which arecapable of binding to the antigen. Fab and F(ab′)₂ fragments lack the Fcfragment of intact antibody, clear more rapidly from the circulation andmay have less non-specific tissue binding than an intact antibody (28).Such fragments are typically produced by proteolytic cleavage, usingenzymes such as papain (to generate Fab fragments) and pepsin (togenerate F(ab′)₂ fragments). It will be appreciated that Fab and F(ab′)₂and other fragments of the antibodies useful in the present inventionmay be used for the detection or quantitation of GP88, and for treatmentof pathological states related to GP88 expression, according to themethods disclosed herein for intact antibody molecules.

According to the present invention, antibodies that neutralize GP88activity in vitro can be used to neutralize GP88 activity in vivo totreat diseases associated with increased GP88 expression or increasedresponsiveness to GP88, such as but not limited to cancer and viralinfection. A subject, preferably a human subject, suffering from diseaseassociated with increased GP88 expression is treated with an antibody toGP88. Such treatment may be performed in conjunction with otheranti-cancer or anti-viral therapy. A typical regimen comprisesadministration of an effective amount of the antibody specific for GP88administered over a period of one or several weeks and including betweenabout one and six months. The antibody of the present invention may beadministered by any means that achieves its intended purpose. Forexample, administration may be by various routes including but notlimited to subcutaneous, intravenous, intradermal, intramuscular,intraperitoneal and oral. Parenteral administration can be by bolusinjection or by gradual perfusion over time. Preparations for parenteraladministration include sterile aqueous or non-aqueous solutions,suspensions and emulsions, which may contain auxiliary agents orexcipients known in the art. Pharmaceutical compositions such as tabletsand capsules can also be prepared according to routine methods. It isunderstood that the dosage of will be dependent upon the age, sex andweight of the recipient, kind of concurrent treatment, if any, frequencyof treatment and the nature of the effect desired. The ranges ofeffective doses provided below are not intended to limit the inventionand merely represent preferred dose ranges. However the most preferreddosage will be tailored to the individual subject as is understood anddeterminable by one skilled in the art. The total dose required for eachtreatment may be administered by multiple doses or in a single dose.Effective amounts of antibody are from about 0.01 μg to about 100 mg/kgbody weight and preferably from about 10 μg to about 50 mg/kg. Antibodymay be administered alone or in conjunction with other therapeuticsdirected to the same disease.

According to the present invention and concerning the neutralizingantibody, GP88 neutralizing antibodies can be used in all therapeuticcases where it is necessary to inhibit GP88 biological activity, eventhough there may not necessarily be a change in GP88 expression,including cases where there is an overexpression of GP88 cell surfacereceptors and this in turn results in an increased biological activity,or where there is an alteration in GP88 signaling pathways or receptorsleading to the fact that the signaling pathways are always “turned on.”Neutralizing antibodies to growth factor and to growth factor receptorshave been successfully used to inhibit the growth of cells whoseproliferation is dependent on this growth factor. This has been the casefor IGF-I receptor in human breast carcinoma cells (14) and bombesin forlung cancer (29). The antibody to GP88 can also be used to delivercompounds such as, but not limited to, cytotoxic reagents such astoxins, oncotoxins, mitotoxins and immunotoxins, or antisenseoligonucleotides, in order to specifically target them to cellsexpressing or responsive to GP88 (30).

One region that allows antigen to develop a neutralizing antibody toGP88 is the 19 amino-acid region defined as K19T in the mouse GP88, andE19V in the human GP88 which is not located within theepithelin/granulin 6 kDa repeats but between these repeats, specificallybetween granulin A (epithelin 1) and granulin C (5) in what isconsidered a variant region (see FIG. 10). Without wishing to be boundby theory, it is believed that the region important for the biologicalactivity of GP88 lies outside of the epithelin repeats.

The antibodies or fragments of antibodies useful in the presentinvention may also be used to quantitatively or qualitatively detect thepresence of cells which express the GP88 protein. This can beaccomplished by immunofluorescence techniques employing a fluorescentlylabeled antibody (see below) with fluorescent microscopic, flowcytometric, or fluorometric detection. The reaction of antibodies andpolypeptides of the present invention may be detected by immunoassaymethods well known in the art (20).

The antibodies of the present invention may be employed histologicallyas in light microscopy, immunofluorescence or immunoelectron microscopy,for in situ detection of the GP88 protein in tissues samples orbiopsies. In situ detection may be accomplished by removing ahistological specimen from a patient and applying the appropriatelylabeled antibody of the present invention. The antibody (or fragment) ispreferably provided by applying or overlaying the labeled antibody (orfragment) to the biological sample. Through the use of such a procedure,it is possible to determine not only the presence of the GP88 proteinbut also its distribution in the examined tissue. Using the presentinvention, those of ordinary skill in the art will readily perceive thatany wide variety of histological methods (such as staining procedures)can be modified in order to achieve such in situ detection.

Assays for GP88 typically comprise incubating a biological sample suchas a biological fluid, a tissue extract, freshly harvested or culturedcells or their culture medium in the presence of a detectably labeledantibody capable of identifying the GP88 protein and detecting theantibody by any of a number of techniques well known in the art.

The biological sample may be treated with a solid phase support orcarrier such as nitrocellulose or other solid support capable ofimmobilizing cells or cell particles or soluble proteins. The supportmay then be washed followed by treatment with the detectably labeledanti-GP88 antibody. This is followed by wash of the support to removeunbound antibody. The amount of bound label on said support may then bedetected by conventional means. By solid phase support is intended anysupport capable of binding antigen or antibodies such as but not limitedto glass, polystyrene polypropylene, nylon, modified cellulose, orpolyacrylamide.

The binding activity of a given lot of antibody to the GP88 protein maybe determined according to well known methods. Those skilled in the artwill be able to determine operative and optimal assay conditions foreach determination by employing routine experimentation.

Detection of the GP88 protein or functional derivative thereof and of aspecific antibody for the protein may be accomplished by a variety ofimmunoassays well known in the art such as enzyme linked immunoassays(EIA) or radioimmunoassays (RIA). Such assays are well known in the artand one of skill will readily know how to carry out such assays usingthe anti-GP88 antibodies and GP88 protein of the present invention.

Such immunoassays are useful to detect and quantitate GP88 protein inserum or other biological fluid as well as in tissues, cells, cellextracts, or biopsies. In a preferred embodiment, the concentration ofGP88 is measured in a tissue specimen as a means for diagnosing canceror other disease associated with increased expression of GP88.

The presence of certain types of cancers and the degree of malignancyare said to be “proportional” to an increase in the level of the GP88protein. The term “proportional” as used herein is not intended to belimited to a linear or constant relationship between the level ofprotein and the malignant properties of the cancer. The term“proportional” as used herein, is intended to indicate that an increasedlevel of GP88 protein is related to appearance, recurrence or display ofmalignant properties of a cancer or other disease associated withincreased expression of GP88 at ranges of concentration of the proteinthat can be readily determined by one skilled in the art.

Another embodiment of the invention relates to evaluating the efficacyof anti-cancer or anti-viral drug or agent by measuring the ability ofthe drug or agent to inhibit the expression or production of GP88. Theantibodies of the present invention are useful in a method forevaluating anti-cancer or anti-viral drugs in that they can be employedto determine the amount of the GP88 protein in one of theabove-mentioned immunoassays. Alternatively, the amount of the GP88protein produced is measured by bioassay (cell proliferation assay) asdescribed herein. The bioassay and immunoassay can be used incombination for a more precise assessment.

An additional embodiment is directed to an assay for diagnosing cancersor other diseases associated with an increase in GP88 expression basedon measuring in a tissue or biological fluid the amount of mRNAsequences present that encode GP88 or a functional derivative thereof,preferably using an RNA-DNA hybridization assay. The presence of certaincancers and the degree of malignancy is proportional to the amount ofsuch mRNA present. For such assays the source of mRNA will be biopsiesand surrounding tissues. The preferred technique for measuring theamount of mRNA is a hybridization assay using DNA of complementaritybase sequence.

Another related embodiment is directed to an assay for diagnosingcancers or other diseases associated with an increase in GP88responsiveness based on measuring on a tissue biopsy whether treatmentwith anti-GP88 neutralizing antibody will inhibit its growth or otherbiological activity.

Another related embodiment is a method for measuring the efficacy ofanti-cancer or anti-viral drug or agent which comprises the steps ofmeasuring the agent's effect on inhibiting the expression of mRNA forGP88. Similarly such method can be used to identify or evaluate theefficacy of GP88 antagonizing agents by measuring the ability of saidagent to inhibit the production of GP88 mRNA.

Nucleic acid detection assays, especially hybridization assays, can bebased on any characteristic of the nucleic acid molecule such as itssize, sequence, or susceptibility to digestion by restrictionendonucleases. The sensitivity of such assays can be increased byaltering the manner in which detection is reported or signaled to theobserver. A wide variety of labels have been extensively developed andused by those of ordinary skill in the art, including enzymatic,radioisotopic, fluorescent, chemical labels and modified bases.

One method for overcoming the sensitivity limitation of a nucleic acidfor detection is to selectively amplify the nucleic acid prior toperforming the assay. This method has been referred as the “polymerasechain reaction” or PCR (31 and U.S. Pat. Nos. 4,683,202 and 4,582,788).The PCR reaction provides a method for selectively increasing theconcentration of a particular nucleic acid sequence even when thatsequence has not been previously purified and is present only in asingle copy in a particular sample.

GP88 Antisense Components

This invention also provides GP88 antisense components. The constitutiveexpression of antisense RNA in cells has been shown to inhibit theexpression of more than 20 genes and the list continues to grow (32-34).Possible mechanisms for antisense effects are the blockage oftranslation or prevention of splicing, both of which have been observedin vitro. Interference with splicing allows the use of intron sequences(30) which should be less conserved and therefore result in greaterspecificity, inhibiting expression of a gene product of one species butnot its homologue in another species.

The term antisense component corresponds to an RNA sequence as well as aDNA sequence coding therefor, which is sufficiently complementary to aparticular mRNA molecule, for which the antisense RNA is specific, tocause molecular hybridization between the antisense RNA and the mRNAsuch that translation of the mRNA is inhibited. Such hybridization canoccur under in vivo conditions. The action of the antisense RNA resultsin specific inhibition of gene expression in the cells (32-35).

According to the present invention, transfection of tumorigenic cellswith DNA antisense to the GP88 cDNA inhibits endogenous GP88 expressionand inhibits tumorigenicity of the antisense cDNA transfected cells.This antisense DNA must have sufficient complementarity, about 18-30nucleotides in length, to the GP88 gene so that the antisense RNA canhybridize to the GP88 gene (or mRNA) and inhibit GP88 gene expressionregardless of whether the action is at the level of splicing,transcription, or translation. The degree of inhibition is readilydiscernible to one skilled in the art without undue experimentationgiven the teachings herein and preferably is sufficient to inhibit thegrowth of cells whose proliferation is dependent on the expression ofGP88. One of ordinary skill in the art will recognize that the antisenseRNA approach is but a number of known mechanisms which can be employedto block specific gene expression.

The antisense components of the present invention may be hybridizable toany of several portions of the target GP88 cDNA, including the codingsequence, 3′ or 5′ untranslated regions, or other intronic sequences, orto GP88 mRNA. As is readily discernible by one of ordinary skill in theart, the minimal amount of homology required by the present invention isthat sufficient to result in hybridization to the GP88 DNA or mRNA andin inhibition of transcription of the DNA, or translation or function ofthe mRNA, preferably without affecting the function of other mRNAmolecules and the expression of other unrelated genes.

Antisense RNA is delivered to a cell by transformation or transfectionvia a vector, including retroviral vectors and plasmids, into which hasbeen placed DNA encoding the antisense RNA with the appropriateregulatory sequences including a promoter to result in expression of theantisense RNA in a host cell. Stable transfection of various antisenseexpression vectors containing GP88 cDNA fragments in the antisenseorientation have been performed. One can also deliver antisensecomponents to cells using a retroviral vector. Delivery can also beachieved by liposomes.

For purpose of antisense technology for in vivo therapy, the currentlypreferred method is to use antisense oligonucleotides (32, 36), insteadof performing stable transfection of an antisense cDNA fragmentconstructed into an expression vector. Antisense oligonucleotides havinga size of 15-30 bases in length and with sequences hybridizable to anyof several portions of the target GP88 cDNA, including the codingsequence, 3′ or 5′ untranslated regions, or other intronic sequences, orto GP88 mRNA, are preferred. Sequences for the antisenseoligonucleotides to GP88 are preferably selected as being the ones thathave the most potent antisense effects (37, 38). Factors that govern atarget site for the antisense oligonucleotide sequence are related tothe length of the oligonucleotide, binding affinity, and accessibilityof the target sequence. Sequences may be screened in vitro for potencyof their antisense activity by measuring inhibition of GP88 proteintranslation and GP88 related phenotype, e.g., inhibition of cellproliferation in cells in culture. In general it is known that mostregions of the RNA (5′ and 3′ untranslated regions, AUG initiation,coding, splice junctions and introns) can be targeted using antisenseoligonucleotides.

The preferred GP88 antisense oligonucleotides are those oligonucleotideswhich are stable, have a high resilience to nucleases (enzymes thatcould potentially degrade oligonucleotides), possess suitablepharmacokinetics to allow them to traffic to disease tissue at non-toxicdoses, and have the ability to cross through plasma membranes.

Phosphorothioate antisense oligonucleotides may be used (39).Modifications of the phosphodiester linkage as well as of theheterocycle or the sugar may provide an increase in efficiency. Withrespect to modification of the phosphodiester linkage, phosphorothioatemay be used. An N3′-P5′ phosphoramidate linkage has been described asstabilizing oligonucleotides to nucleases and increasing the binding toRNA (40). Peptide nucleic acid (PNA) linkage is a complete replacementof the ribose and phosphodiester backbone and is stable to nucleases,increases the binding affinity to RNA, and does not allow cleavage byRNAse H. Its basic structure is also amenable to modifications that mayallow its optimization as an antisense component. With respect tomodifications of the heterocycle, certain heterocycle modifications haveproven to augment antisense effects without interfering with RNAse Hactivity. An example of such modification is C-5 thiazole modification.Finally, modification of the sugar may also be considered. 2′-O-propyland 2′-methoxyethoxy ribose modifications stabilize oligonucleotides tonucleases in cell culture and in vivo. Cell culture and in vivo tumorexperiments using these types of oligonucleotides targeted to c-raf-1resulted in enhanced potency. As general references for antisenseoligonucleotides, see (32-34)

The delivery route will be the one that provides the best antisenseeffect as measured according to the criteria described above. In vitrocell culture assays and in vivo tumor growth assays using antisenseoligonucleotides have shown that delivery mediated by cationicliposomes, by retroviral vectors and direct delivery are efficient. (36,41-43) Another possible delivery mode is targeting using antibody tocell surface markers for the tumor cells. Antibody to GP88 or to itsreceptor may serve this purpose.

Recombinant GP88

The present invention is also directed to DNA expression systems forexpressing a recombinant GP88 polypeptide or a functional derivativethereof substantially free of other mammalian DNA sequences. Such DNAmay be double or single stranded. The DNA sequence should preferablyhave about 20 or more nucleotides to allow hybridization to anotherpolynucleotide. In order to achieve higher specificity of hybridization,characterized by the absence of hybridization to sequences other thanthose encoding the GP88 protein or a homologue or functional derivativethereof, a length of at least 50 nucleotides is preferred.

The present invention is also directed to the above DNA molecules,expressible vehicles or vectors as well as hosts transfected ortransformed with the vehicles and capable of expressing the polypeptide.Such hosts may be prokaryotic, preferably bacteria, or eukaryotic,preferably yeast or mammalian cells. A preferred vector system includesbaculovirus expressed in insect cells. The DNA can be incorporated intohost organisms by transformation, transduction, transfection, infectionor related processes known in the art. In addition to DNA and mRNAsequences encoding the GP88 polypeptide, the invention also providesmethods for expression of the nucleic acid sequence. Further, thegenetic sequences and oligonucleotides allow identification and cloningof additional polypeptides having sequence homology to the polypeptideGP88 described here.

An expression vector is a vector which (due to the presence ofappropriate transcriptional and/or translational control sequences) iscapable of expressing a DNA (or cDNA) molecule which has been clonedinto the vector and thereby produces a polypeptide or protein.Expression of the cloned sequence occurs when the expression vector isintroduced into an appropriate host cell. If a prokaryotic expressionvector is employed, then the appropriate host cell would be anyprokaryotic cell capable of expressing the cloned sequence. Similarly,if an eukaryotic expression system is employed, then the appropriatehost cell would be any eukaryotic cell capable of expressing the clonedsequence. Baculovirus vector, for example, can be used to clone GP88cDNA and subsequently express the cDNA in insect cells.

A DNA sequence encoding GP88 polypeptide or its functional derivativesmay be recombined with vector DNA in accordance with conventionaltechniques including blunt-ended or staggered ended termini forligation, restriction enzyme digestion to provide appropriate termini,filling in cohesive ends as appropriate, alkaline phosphatase treatmentto avoid undesirable joining, and ligation with proper enzyme ligases.Techniques for such manipulations are discussed in (35).

A nucleic acid molecule is capable of expressing a polypeptide if itcontains nucleotide sequences which contain transcriptional andtranslational regulatory information and such sequences are operablylinked to nucleotide sequences which encode the polypeptide. An operablelinkage is a linkage in which the regulatory DNA sequences and the DNAsequence sought to be expressed are connected in such a way as to permitgene expression. The precise nature of the regulatory regions needed forgene expression may vary from organism to organism but shall in generalinclude a promoter region, which in prokaryotes contains both thepromoter (which directs the initiation of RNA transcription) as well asthe DNA sequences which when transcribed into RNA will signal theinitiation of protein synthesis. Such regions will normally includethose 5′ non-coding sequences involved with the initiation oftranscription, translation such as the TATA box, capping sequence, CAATsequence and the like.

If desired, the 3′ non-coding region to the gene sequence encoding theprotein may be obtained by described methods (screening appropriate cDNAlibrary or PCR amplification). This region may be retained for thepresence of transcriptional termination regulatory sequences such astermination and polyadenylation. Thus, by retaining the 3′ regionnaturally contiguous to the DNA sequence coding for the protein, thetranscriptional termination signals may be provided. Where thetranscription termination signals are not provided or satisfactorilyfunctional in the expression host cells, then a 3′ region from anothergene may be substituted.

Two DNA sequences such as a promoter region sequence and GP88 encodingsequence are said to be operably linked if the nature of the linkagebetween the sequences does not (1) result in the introduction of aframe-shift mutation or (2) interfere with the ability of the promotersequence to direct transcription of the polypeptide gene sequence.

The promoter sequences may be prokaryotic, eukaryotic or viral. Suitablepromoters are inducible, repressible or constitutive. Examples ofsuitable prokaryotic promoters are reviewed by (44-46).

Eukaryotic promoters include but are not limited to the promoter for themouse methallothionein I gene (47), the TK promoter of Herpes Virus(48), the gene gal4 promoter (49), the SV40 early promoter (50), themouse mammary tumor virus (MMTV) promoter, and the cytomegalovirus (CMV)promoter (51). Strong promoters are preferred. Examples of suchpromoters are those which recognize the T3, SP6 and T7 polymerases, thePL promoter of bacteriophage lambda, the recA promoter, the promoter ofthe mouse methallothionein I gene, the SV40 promoter and the CMVpromoter.

It is to be understood that application of the teachings of the presentinvention to a specific problem or environment will be within thecapability of one having ordinary skill in the art in light of theteachings contained herein. The present invention is more fullyillustrated by the following non-limiting examples.

EXAMPLE 1 Isolation of PC Cell Line

The role of autocrine growth factor production in the loss ofdifferentiation ability and acquisition of tumorigenic properties inmammalian cells has been studied using a murine model system developedby the inventor. It consists of the mouse C3H adipogenic cell line 1246(9), a series of cell lines which are differentiation-deficient and haveincreasing tumorigenic properties. 1246 cells proliferate anddifferentiate in a serum-free defined medium (9). In defined medium,1246 cells stringently require insulin for proliferation and fordifferentiation (10). Insulin-like growth factor I (IGF-I) can replaceinsulin for proliferation but not for differentiation. From 1246 cellsmaintained in the absence of insulin, insulin-dependent cell lines wereisolated (11).

1246-3A was particularly studied. 1246-3A cells had lost the ability todifferentiate and had become tumorigenic in vivo (11). 1246-3A cellsformed tumors when 10⁶ cells were injected into syngeneic C3H micewithin 6 weeks, whereas 1246 cells were non-tumorigenic. By an invitro-in vivo shuttle technique, highly tumorigenic insulin-independentcell lines were subsequently isolated and analyzed (12).

The shuttle technique consisted of subcutaneously injecting 1246-3Acells (10⁶ cells/mouse) into syngeneic C3H mice. The tumor resultingfrom the injection of the cells was then minced and plated in primaryculture into defined medium deprived of insulin (DME-F12 nutrient medium1:1 mixture supplemented with fibronectin, transferrin and FGF). Cellsthat had started to grow were subcultured when they reached confluencyto be: (1) either frozen in the presence of 10% Fetal Bovine serum and10% Dimethylsulfoxyde (DMSO) for long term conservation; (2) injectedsubcutaneously into C3H mice at different cell densities (10⁶, 10⁵, 10⁴cells/mouse). Rate of appearance of tumor and size of tumor wasmonitored. Tumors that appeared were again put back in culture in theinsulin-free medium. Cells growing in these conditions were reinjectedback into the animal.

By this in vitro-in vivo shuttle technique insulin-independent cellswith increasing tumorigenic properties were obtained. In particular, thehighly tumorigenic cell line named PC was isolated (12). PC cells canform tumors even when 10⁴ cells are injected subcutaneously intosyngeneic C3H mice.

The PC cell line has at least the following characteristics:

-   -   (1) These cells represent an excellent model system for        tumorigenicity studies: these cells can proliferate in a simple        defined medium DME-F12 nutrient mixture supplemented with 2        μg/ml fibronectin and 10 μg/ml transferrin, and they can be        injected into syngeneic hosts and do not require the use of nude        mice which are expensive and necessitate special handling.    -   (2) When the PC cells reach confluency, the cells can be        maintained in a complete serum-free and factor-free medium.        Their growth is maintained solely by the nutrient medium and the        factors that the cells secrete in their conditioned medium, thus        conditioned medium is a good source for characterization and        purification of factors required for proliferation of tumor        cells.

EXAMPLE 2 GP88 is an Autocrine Growth Factor for the Highly TumorigenicPC Cells

It was shown that PC cell conditioned medium contained growth promotingactivity that was purified by chromatographic techniques (4). Thepurified factor called GP88 precursor was sequenced and shown to besimilar to the epithelin/granulin precursor.

Experiments were then carried out to examine whether the production ofGP88 by PC cells stimulated their growth in an autocrine fashion. Forthis purpose, PC cells were cultivated in the presence of GP88 antibodythat can neutralize GP88 activity. DNA synthesis of PC cells wasmeasured in the presence of increasing amounts of either the non-immuneIgG or the anti-K19T IgG.

As shown in FIG. 2, the addition of anti-GP88 IgG inhibited PC cellgrowth in a dose-dependent manner, directly demonstrating that GP88production by the PC cells is required for their growth. Non-immune IgGhad no effect. Here, PC cells were plated in 96-well plates at a densityof 2×10⁴ cells/well in DME/F12 medium supplemented with 2 μg/mlfibronectin and 10 μg/ml transferrin (2F medium). After 6 hours when thecells were attached, anti-GP88 IgG fraction was added. 36 hours later,³H-thymidine (0.25 μCi/ml) was added for an additional 8 hours. Cellswere collected by trypsinization on glass filters by a cell harvesterand the radioactivity corresponding to ³H-thymidine incorporated intoDNA counted by liquid scintillation counter. Values (FIG. 2) areexpressed as % of control corresponding to thymidine incorporation in PCcells treated with equivalent amounts of nonimmune IgG.

Similar results were obtained using anti K19T and anti E19V monoclonalantibodies. Anti K19T monoclonal antibody inhibited the growth of PCcells and of rat leukemia cells in a dose dependent fashion. Moreover,anti E19V monoclonal antibody inhibited the growth of the human breastcarcinoma cell line MCF7, with an ED₅₀ of 100 μg/ml.

EXAMPLE 3 Expression of GP88 in the 1246, 1246-3A and PC Cell Lines

Since GP88 protein was purified from PC cell conditioned medium,experiments were carried out to compare the expression of GP88 mRNA andprotein in the three cell lines.

Comparative tumorigenicity studies of 1246, 1246-3A and PC cell lines inC3H mice showed that PC cells are highly tumorigenic when compared to1246-3A cells since they can develop tumors when 10⁴ cells/mouse areinjected into C3H mice. 1246-3A cells make tumors when injected a 10⁶cells/mouse, whereas in syngeneic hosts, 1236 cells are non-tumorigenic(12).

The following methods were used for the studies of comparing the levelof GP88 in the model system consisting of the three cell lines 1246,1246-3A and PC.

Cell Culture

1246 stock cells were maintained in DME/F12 nutrient medium (1:1 mixtureof Dulbecco's modified Eagle medium and Ham's nutrient F12) supplementedwith 10% fetal bovine serum (FBS). 1246-3A and PC stock cells weremaintained in defined media. For PC stock cells, it consisted of DME-F12medium supplemented with 2 μg/ml of human plasma fibronectin and 10μg/ml of human plasma transferrin (2F medium). For 1246-3A cells, thedefined medium called 3F medium consisted of DME-F12 medium supplementedwith 2 μg/ml of human plasma fibronectin and 10 μg/ml of human plasmatransferrin and 1 ng/ml of basic fibroblast growth factor (bFGF). Forcomparative studies the three cell lines were plated in DME-F12 mediumsupplemented with 2% FBS.

RNA Isolation and Northern Blot Analysis

For Northern blot analysis of GP88 mRNA expression in rodent cells ortissues (mouse and rats), we used a mouse GP88 cDNA probe 311 bp inlength starting at nucleotide 551 to 862 (corresponding to amino-acidsequence 160 to 270). The probe was ³²P-labeled by random-primingreaction.

Total cellular RNA was isolated by RNAZOL solution (Cinnabiotech) orTRIZOL solution (Life Technologies) based on a modification of thesingle step guanidinium isothiocyanante/phenol chloroform method (52).

Fifteen or twenty micrograms of total RNA per sample were subjected toelectrophoresis on a denaturing 1.2% agarose gel containing 0.22 Mformaldehyde in 1×MOPS (10×MOPS: 0.2 M MOPS, 50 mM NaGAc 10 mM EDTA).RNA was blotted on nitrocellulose membrane (MSI Inc., Westboro, Mass.)by overnight capillary transfer in 10×SSC (20×SSC =3M NaCl, 0.3M NaCitrate pH 7.0). The filters were baked at 80° C. under vacuum for 2 hrsand then prehybridized at 42° C. for 4 hrs in hybridization solutionconsisting of 50% formamide, 5×SSPE (1×SSPE =0.16 M sodium chloride, 50mM sodium phosphate pH 7.4, 1 mM EDTA), 1% SDS, 5×Denhardt's solution(1×Denhardt's solution =0.02 % each of polyvinylpirrolidone, Ficoll andbovine serum albumin), 1 μg/ml poly-A and 100 μg/ml denatured salmonsperm DNA at 42° C. Hybridization was performed overnight at 42° C. inthe same solution with 10⁶cpm/ml of random-primed ³²P-labeled GP88 cDNAprobe. Filters were washed twice for 25 min at 42° C. in 2×SSC and 1%SDS, followed by two 15 mm. washes at 56° C. in 0.2×SSC and 1% SDS.Dried filters were exposed to KODAK XAR-5 film (Kodak, Rochester, N.Y.)at -70° C. with an intensifying screen (Dupont, Boston, Mass.). Resultswere quantitated by densitometric scanning. Ribosomal protein L₃₂ mRNAwas detected as internal standard for normalizing RNA loading.

Preparation of Cell Lysate, Immunoprecipitation and Western BlotAnalysis

Cells in 10 cm culture dishes were washed once with PBS buffer and lysedon ice for 10 min. with 1 ml PBS buffer containing 1% Triton X-100. Celllysate was sonicated for 10 seconds on ice, centrifuged at 10,000×g for10 min., then the supernatant was collected and stored at −70° C. untiluse.

Amounts of cell lysate and culture medium to be analyzed were normalizedby cell number of 18×10⁵ and 3×10⁵ respectively. The proteaseinhibitors: 200 μM PMSF, 1 μM leupeptin, 0.5 μM aprotinin, and 1 μM EDTAwere added per sample. Each sample was incubated at 4° C. for 4 hourswith 5 μg of affinity purified anti-K19T IgG conjugated to agarose withshaking. Precipitates were collected by centrifugation, washed threetimes with PBS buffer, resuspended in 20 μl SDS sample buffer containing5% β-mercaptoethanol, boiled for 5 min., and then separated by SDS-PAGEon 10% polyacrylamide gels according to the method of Laemmli (56).Proteins were electro transferred to immobilon membranes and GP88detected using anti K19T antibody conjugated to horseradish peroxidaseand detected by enhanced chemiluminescence (ECL).

When expression of GP88 mRNA was investigated in the three cell linescultivated in DME/F12 medium supplemented with 2% fetal bovine serum(FBS) our laboratory as a probe the results show that the highest levelof mRNA expression for GP88 is found in the highly tumorigenic PC cells(FIG. 1B).

The levels of GP88 protein expression were examined by Western blotanalyses using anti-K19 antibody both in cell lysates and in culturemedium of 1246, 1246-3A and PC cells as described above. As shown inFIG. 1A, the level of GP88 was undetectable in the culture medium of1246 cells, 3T3, and 1246-3A cells and increased dramatically in theculture medium of the highly tumorigenic PC cells. The same results wereobtained for GP88 expression in cell lysates.

GP88 expression was examined in 1246 cells in different cultureconditions such as defined medium and serum containing medium. It wasshown that the level of expression of GP88 mRNA (measured by Northernblot analysis) and protein (measured by Western blot analysis) in 1246and in 1246-3A cells is inhibited when the cells are treated with 2%fetal bovine serum indicating the presence of circulating inhibitors ofGP88 expression in fetal bovine serum (FIG. 1C). This inhibition of GP88expression was also observed when the activity of GP88 promoter linkedto a luciferase reporter gene was measured indicating that theseinhibitors are effective in inhibiting the transcriptional activity ofthe GP88 gene. Such inhibitors can be useful to develop GP88antagonizing agents which will be useful as anti-tumor or antiviraltherapy. We have also showed that GP88 mRNA expression is stimulated byEGF in the 1246 cells and by insulin in the human mammary carcinomacells MDA MB-453.

EXAMPLE 4 GP88 Expression and Biological Activity in Mammary EpithelialCells

(a) Expression of GP88 in Breast Carcinoma Cells.

Experiments were carried out to examine the level of GP88 expression inbreast carcinoma and to examine if GP88 is a growth factor for mammaryepithelial cells. The rationale in support of this possibility is: GP88is a potent growth stimulator for mammary epithelial cells (see nextparagraph); receptors for GP88 (our studies) and processed formepithelin 1 (54) have been characterized on mammary epithelial cells;breast carcinoma cell lines with different degrees of hormonaldependency are available for study; and lastly there are a growingnumber of reports emphasizing the importance of the insulin/IGF pathwayin the growth control of mammary cells indicating that an escape fromthis regulation is occurring in malignant breast carcinomas (14, 15).Since PC cells which display an over expression of GP88 are insulin/IGFindependent, this would support the rationale of GP88 deregulation inhuman breast carcinoma.

We have investigated the level of expression of GP88 mRNA in three wellstudied human breast carcinoma cell lines, MCF-7, which is estrogenreceptor positive (ER+), and two cell lines, MDA-MB-453 and 468, beingestrogen receptor negative (ER−). MDA-MB-468 has also been characterizedas having a defective insulin and insulin-like growth factor signaling(53). FIG. 5 shows that GP88 mRNA is expressed in the three cell linesbut that the level of expression is higher in the ER− cell lines,MDA-MB-453 and 468, than in the ER+MCF7 cells, indicating that in humanbreast carcinoma, increased malignancy may be accompanied by increasedGP88 expression.

(b) Biological Activity of GP88 in Mammary Epithelial Cells

We investigated the effect of GP88 on the proliferation of a variety ofcell lines including fibroblasts and mammary epithelial cells. We havefound that GP88 had a profound growth promoting effect on the mousemammary epithelial cell line C57MG. As shown in FIG. 6, a 5-foldincrease in DNA synthesis was observed at a concentration of 150 ng/ml(2 nM) either with GP88 purified from PC cells or with recombinant GP88expressed in insect cells.

The ability of GP88 purified from PC cells (upper panel) and recombinantGP88, rGP88 (lower panel), to stimulate DNA synthesis in C57MG cells wasmeasured by incorporation of ³H-thymidine in serum-starved quiescentcells. Interestingly, in contrast to the growth promoting effect of GP88on breast epithelial cells, the 6 kDa epithelin 2 (epi 2) has beenreported as a growth inhibitor, at least for MDA-MB-468 cells when givenat concentrations up to 100 nM (54). These data suggest that theprecursor, i.e., GP88, and the processed form, i.e., epi 2, haveopposite effects on mammary epithelial cell growth.

EXAMPLE 5 Cloning of GP88 cDNA

GP88 protein purified from PC cell conditioned medium was sequencedafter digestion with cyanogen bromide and trypsin. Sequences ofN-terminal regions and 6 peptides were obtained (4). Sense and antisenseredundant oligonucleotide primers complementary to the obtained aminoacid sequences were synthesized and used in the polymerase chainreaction using the touch down PCR method with first strand cDNA of PCcells as template. From the touch down PCR using a primer pair SCV157and ANG300, a 444 bp amplified product was obtained. This cDNA was thenused to screen a lambda-ZAP cDNA library prepared from PC cells in ourlaboratory. One million unamplified plaques were screened by plaquehybridization with ³²P-labeled PCR generated cDNA fragment. Positiveclones were isolated by an additional 3 rounds of plaque purification.Full length GP88 cDNA clone was obtained and sequenced. Full length cDNAwas 2137 nucleotides in length with the first ATG located at 23 bp fromthe 5′ end, an open reading frame (ORF) 1770 nucleotides long, and a3′untranslated region having a polyA tail at position 2127. The sequencewas identical to the published mouse granulin (5) except for onenucleotide (T instead of G) at position 1071 of GP88 cDNA (position 1056of mouse granulin), which resulted in the change of amino acid fromarginine to leucine, and a nucleotide substitution at position 1483 withno change in amino acid (FIG. 8). This study demonstrated that GP88 issimilar to epithelin/granulin precursor and provided a cDNA to pursueour study of GP88 expression.

EXAMPLE 6 Expression of Mouse GP88 cDNA in Baculovirus

For recombinant GP88 production, the method of choice was to expressGP88 in the baculovirus system. A full length GP88 cDNA (obtained byscreening PC cell cDNA library) including the signal peptide was ligatedinto the baculovirus transfer vector pVL1392 (in Vitrogen, San Diego,Calif.). Plasmid pVL1392-GP88 was used to co-transfect Sf9 insect cellswith baculovirus DNA. Recombinant viruses encoding GP88 were isolatedand plaque purified. For infection and production of recombinant GP88,Sf9 cells were seeded in Grace's medium containing 10% fetal bovineserum (FBS) in T75 cm² flasks. After infection with recombinantbaculovirus-GP88, insect cells were maintained in Grace's medium for 48hours at 27° C. Conditioned medium was collected by centrifugation andrecombinant GP88 (rGP88) was purified by a 2 step purification protocolconsisting of heparin-sepharose and immunoaffinity chromatography asdescribed in Example 6. SDS-PAGE analysis of rGP88 indicated that rGP88migrates faster than PC cell derived GP88 corresponding to an apparentMW of 76 kDa. N-terminal sequencing analysis of rGP88 indicated that itwas identical to GP88 purified from PC-CM. The difference of molecularweight between GP88 and rGP88 is due to a difference in glycosylationstatus of GP88 in insect cells. As shown in FIG. 6, biological activityof rGP88 was identical to that of GP88 purified from PC cells,indicating that the different glycosylation status of GP88 in insectcells and mammalian cells did not affect the biological potency of theprotein.

The rGP88 produced from insect cells can be used for biological, andbinding studies and to develop monoclonal antibodies to the intact GP88

EXAMPLE 7 Purification of GP88 and Recombinant GP88 by AffinityChromatography

The conditioned medium (2000 ml) from PC cells was diluted with the samevolume of H₂O and loaded on a 2.5 ml heparin-sepharose CL-6B columnequilibrated in 10 mM sodium phosphate buffer pH 7.4 containing 75 mMNaCI (Pharmacia, Uppsala, Sweden). The column was washed with at least10 bed volumes of the same equilibration buffer followed by a wash with10 mM sodium phosphate buffer containing 0.15 M NaCi. The fractioncontaining GP88 was eluted with 5 bed volumes of 0.4 M NaCI, 10 mMTris-HCI, pH 7.5. The eluate was stored at −20 C for furtherpurification. A synthetic peptide K19T (SEQ ID NO: 3) (sequence:KKVIAPRRLPDPQILKSDT) was used to raise the antisera against the GP88used in the immunoaffinity step. The K19T peptide was linked toCNBr-activated Sepharose 4B according to the method provided by themanufacturer (Pharmacia, Uppsala, Sweden). The specific anti-K19antibody was purified using the K19T peptide affinity column by elutionat acidic pH. Specifically, anti-K19T IgG was applied to a K19Tpeptide-Sepharose 4B column equilibrated with 10mM sodium phosphatebuffer pH 6.5 (Buffer A) at a flow rate of 0.8 ml/hr, and circulated at4° C. overnight. After washing the column with 7 ml of Buffer A, theconjugate was eluted with 1 ml of HCI, pH 2.9, then 1 ml of HCl, pH 2.5at a flow rate of about 0.1 ml/min in a tube containing 0 1 ml of 1Msodium phosphate buffer pH 7.0 to neutralize the pH. The concentrationof affinity-purified IgG was determined by the absorbance at 280 nm.

The purified Ab-K19T (1 mg) was then conjugated to 1 ml of agarose beads(Sulfolink coupling gel, Pierce, Rockford, Ill.) using protocolsprovided by the manufacturer. The final coupled column contained 600 μganti-K19T/ml gel. The Ab-K19T agarose was packed in a column and washedextensively with PBS. The eluate from heparin sepharose CL-6B column wasdiluted with 3 volumes H₂O and loaded on the Ab-K19T column. Afterwashing the column with buffer consisting of 750 mM NaCl in 10 mM NaPO4pH 7.5, the fraction containing GP88 was eluted by elution buffer (150mM NaCl, pH 2.5 (HCl)). To neutralize, 1/10 volume (v/v) 1 M sodiumphosphate pH 6.5 buffer was added to the eluate and the proteinconcentration was determined by amino acid analysis or micro BCA kit(Bio-Rad, Richmond, Calif.). In general 50 μg of GP88 was purified on a350 μl column.

This method is also adequate for the purification of recombinant GP88such as constructed in a baculovirus expression vector and expressed ininsect cells. This method is also adequate to purify human GP88 usingfor the immunoaffinity step human GP88 antibody conjugated to adequatesupport (sepharose or agarose).

EXAMPLE 8 Development of Neutralizing Antibody for GP88

Peptides corresponding to various regions of mouse and human GP88 weresynthesized and conjugated to keyhole limpet Hemocyanin (KLH) by the“glutaraldehyde method. ” Peptide KLH conjugate was injected intochinchilla rabbits to raise anti-GP88 antibody. Two peptides, K19T andS14R, listed below, were found to generate neutralizing antibodies.Equivalent regions such as E19V of the human GP88 amino acid sequenceswere used to develop neutralizing anti-human GP88 monoclonal antibodies.Peptides were as follows:

P12T (SEQ ID NO: 4) PDAKTQCPDDST from P208 to T219 K19T (SEQ ID NO: 3)KVIAPRRLPDPQILKSDT from K344 to T362 S14R (SEQ ID NO: 5) SARGTKCLRKKIPRfrom S562 to R575 E19V (SEQ ID NO: 6) EKAPAHLSLPDPQALKRDV (human GP88)A14R (SEQ ID NO: 7) ARRGTKCLRREAPR (human GP88)

Properties of anti-sera are the following:

(1) Anti-K19T, anti-S14R and anti-E19V antibody recognize an 88 kDa GP88in conditioned medium of cells in culture, in cell lysates and in tissueextracts.

Tissue distribution of GP88 protein expression indicates that it iswidely expressed and that most tissues express the unprocessedprecursor, i.e., GP88, rather than processed 6 kDa forms, i.e., epi 1and 2. GP88 is found in serum (mouse serum contains about 150 ng 88 kDaGP88/ml), and expressed in adipose tissue, in brain (molecular weightvaries between 45 and 88 kDa), in testes, in ovary, in liver and inkidney.

(2) Anti-K19T antibody is a neutralizing antibody. Anti-K19T antibodyneutralizes the biological effect of GP88 secreted by PC cells which isrequired for their proliferation. (FIG. 2). Addition of anti-K19T IgGinto culture medium of PC cells results in a dose dependent inhibitionof PC cell growth. Non-immune IgG had no effect. Inhibition of cellproliferation of cells expressing GP88 has also been obtained usingmonoclonal antibody K19T for PC cells, rat leukemia cell lines and usingmonoclonal antibody E19V for neutralizing human GP88 in human breastcarcinomas. This demonstrates that the E19V region of human GP88 (andK19T region of mouse GP88) is a region of great biological importanceand that any antibody raised against this region will result inobtaining a neutralizing antibody that can be used for therapy ofdiseases due to increased GP88 expression or increased responsiveness toGP88. The same is true for the S14R region of mouse GP88 and A14R regionof human GP88.

(3) Anti E19V monoclonal antibody is a neutralizing antibody. We haveshown that at a dose of 100 μg/ml, E19V antibody inhibits the growth ofthe human breast carcinoma cell line MCF7 by 50% using ³H-thymidineincorporation assay as described above for PC cells.

EXAMPLE 9 Growth and Tumorigenic Properties of Cells Transfected withExpression Vector Containing GP88 cDNA in Antisense Orientation.

The examples above demonstrate that GP88 is overexpressed by the highlytumorigenic PC cell line. Since the cultivation of PC cells in thepresence of neutralizing anti-GP88 antibody had resulted in growthinhibition, it indicated that GP88 is required for the growth of PCcells. In order to test whether GP88 overexpression is responsible forthe high tumorigenic properties of PC cells in vivo, we examined thegrowth properties and the tumorigenic ability of PC cells transfectedwith a cytomegalovirus promoter controlled expression vector containingGP88 cDNA in antisense orientation in order to obtain high levels ofantisense RNA transcription. As control, we used PC cells transfectedwith empty vector.

A 228 bp fragment of GP88 cDNA was cloned in the antisense orientationin pCMV4 expression vector (Andersson, S., et al, 1989) (51) containingCMV promoter and hGH transcription termination and polyadenylationsignals (pCMV4-GP88AS). PC cells were co-transfected with the 20 μg ofantisense pCMV4-GP88AS and 2 μg of pRSVneo expression vector containingthe neomycin resistant gene by the calcium phosphate method. Controlcells were co-transfected with empty pCMV4 vector and pRSVneo asdescribed above. Transfected cells were selected in the presence ofneomycin. Neomycin resistant colonies were cloned and cells were assayedfirst by detecting the presence of pCMV4-GP88AS by PCR. Twenty-fourpositive neomycin resistant clones containing the antisense pCMV4-GP88ASwere isolated. Nine have been isolated and screened for expression ofthe antisense transcript. Three clones were further characterized.Western blot analysis of cell lysates and conditioned medium usinganti-GP88 antiserum (i.e., anti-K19T antibody) was performed in order todetermine the level of GP88 expression in transfected antisense cellsand control cells (FIG. 7). Culture medium and cell lysates wereprepared by immunoprecipitation with anti-K19T antibody. Protein samplescorresponding to 3×10⁶ cells/lane were analyzed by Western blotting withanti-GP88 antibody. The results indicate that GP88 levels aresignificantly lower in antisense, than in control, transfected cellsparticularly for AS1 and AS18 clones.

Stable Transfection of Antisense GP88 cDNA into PC Cells

PC cells were transfected with a 228 bp antisense cDNA fragment of GP88including start codon region obtained by digesting with Sma I and Xba IGP88 cDNA clone and cloning the obtained cDNA fragment in the antisenseorientation into Xba I and Sma I site of the mammalian expression vectorpCMV4 as shown in FIG. 11. The stable transfection of PC cells wasperformed by the Calcium Phosphate method (55) in DME medium (Dulbecco'sModified eagle Medium) containing 3.7 g/L sodium bicarbonatesupplemented with 10% FBS. 2-4 hours prior to transfection, a calciumphosphate precipitate was made with 20 μg of plasmid pCMV4 constructedwith antisense GP88 cDNA, 2 μof plasmid carrying neomycin resistanceselectable marker (pRSVNEO), and 20 μg of pSK as carrier DNA. After 25minutes, the precipitate was added dropwise to the cells. After 7 hours,the medium was aspirated and the cells were shocked with 10% DMSO in PBSfor 2-3 min., washed twice and fed with complete medium (DMEsupplemented with 10% FBS). One day after transfection, the cells weresplit 1:3 and selected for resistance to Geneticin (G-418 Sulphate,Gibco-BRL) at 400 μg/ml. Media was changed 2 days later to remove deadcells and every 3-4 days thereafter. 10-14 days after the 1:3 split,colonies were picked with a cloning ring and transferred into 48 wellplate, then passaged into 24 well plate, 12 well plate, and 60 mm dishsubsequently. The transfectants were analyzed or frozen. Co-transfectionof the empty pCMV4 vector and pRSVNEO was used for raising the emptyvector control transfectants.

After being selected by their ability to grow in the presence ofGeneticin, the transfected clones were analyzed by two assays asdescribed below:

The presence of the antisense cDNA construct is tested by PCR analysisof genomic DNA of transfected clones using as primers an oligonucleotidelocated in the CMV promoter (SEQ ID NO: 8)(5′-CCTACTTGGCAGTACATCTACGTA-3′) and the other corresponding to thestart codon of GP88 cDNA (SEQ ID NO: 9)(5′-CGAGAATTCAGGCAGACCATGTGGGTC-3′). These primers would amplify a 551bp DNA fragment from genomic DNA of transfected cells containing theantisense DNA construct described above.

Then, the level of GP88 protein in antisense transfected clones wasmeasured by Western blotting analysis of cell lysates and conditionedmedia collected from the transfected clones using anti-K19T antibody tomeasure the efficacy by which the antisense GP88 inhibited theendogenous GP88 protein expression. Antisense clones that showed thehighest degree of inhibition of GP88 expression were selected foranalysis of their growth properties in vivo as described below. Analysisof GP88 expression in empty vector control transfected clones was donesimilarly.

The methods used in these various assays will now be described indetail.

PCR Selection of Transfectants

The presence of the antisense construct in the transfected cells wasdetermined by PCR analysis of their genomic DNA using as sense primerSP647 (SEQ ID NO: 8)(5′-CCTACTTGGCAGTACATCTACGTA-3′) corresponding toCMV promoter region and antisense primer SP7 (SEQ ID NO: 10)(5′-CGAGAAIFfCAGGCAGACCATGTGGGTC-3′) corresponding to start codon regionof GP88. The sense primer SP647 and antisense primer AP912 (SEQ ID NO:11) (5′-CTGACGGTTCACTAAACGAGCTC-3′) both located in the CMV4 promoterwere used to test whether CMV promoter was inserted into the genomic DNAof control transfectants which had been transfected with empty pCMV4vector.

For extracting genomic DNA for the PCR analysis, the transfectants werelysed by buffer A (100 mM KCl, 10 mM Tris-HCl [pH 8.3], 0.45% Tween 20and 0.45% NP40) and proteinase K 120 μg/ml, incubated in 60° C. 1 hr,followed by boiling for 15 min. 50-100 ng DNA of each clone was used astemplate for PCR reaction. Non-transfected PC were used as negativecontrol. Constructed plasmid DNA was used as positive control.

The PCR reaction was performed in 20 μl reaction mixture containing 10mM Tris-HCl pH 9.0, 50 mM KCl, 1.5 mM MgCl2, 0.1% Triton X-100, 0.2 mMdNTPs, 0.5 units Taq DNA polymerase, 3.2 pMol of each primer, and 50-100ng of template DNA. The reaction tubes were heated to 95° C. for 3minutes, and then subjected to 40 cycles of 94° C. for 1 minute, 55° C.for 2 minutes, and 72° C. for 3 minutes with a 10 minutes 72° C.extension in a programmable thermal controller. Products were analyzedon a 1% agarose gel stained with ethidium bromide. The expected size ofthe amplified fragment corresponding to the presence of the antisenseconstruct with the primers chosen was 551 bp.

Measurement of GP88 Protein Expression in Antisense and ControlTransfected Clones

The function of transfecting antisense GP88 DNA in the cells is toinhibit endogenous GP88 expression. Therefore, the transfected clones ofcells which had been selected by the assays described above (i.e.,neomicin resistance and presence of antisense DNA in genomic DNA) wereexamined to determine the degree of inhibition of endogenous GP88expression. This was examined by measuring the level of GP88 in celllysates and in conditioned medium from antisense transfected clones andempty vector transfected control clones by immunoprecipitation andWestern blot analysis using anti-K19T antibody by the methods previouslydescribed. The transfected clones that displayed the highest degree ofinhibition of GP88 expression were further analyzed to examine theirgrowth properties in vitro and in vivo.

Cell Growth Assay

Cells were plated at a density of 3×10⁴ cells/well (12 well plates,Corning) in 2 ml of DME/F-12 medium (1:1 mixture) supplemented with 2%FBS or 2 μg/ml human fibronectin and 10 μg/ml human transferrin. Fivedays later, cells were washed with PBS, and cell number per well wasdetermined by counting cells from duplicate wells using Coulter Counterafter trypsinization.

The results showed that the antisense GP88 transfected PC clones showeda slower growth when cultivated either in serum containing medium and indefined medium (2F medium) than the empty vector control PC cellscultured under equivalent conditions. These results are in agreementwith the fact that GP88 is required by the PC cells to proliferate.Since its expression is inhibited by the antisense, the growth of theantisense transfected cells is inhibited as a result.

Tumorigenicity Assay

Six weeks old female C3H mice were used for tumorigenicity assays.Sense/antisense or control transfectants were injected subcutaneouslyinto syngeneic C3H mice at the density of 10⁶ cells per animal. Theappearance and size of tumors were examined. The mice were killed 45-50days after injection. Tumors were excised, their weight determined andthe tumors were quick frozen and kept at −70° C.

In vivo tumorigenicity studies were are carried out by injecting 5×10⁶antisense and control cells (transfected with empty vector andnontransfected PC cells) subcutaneously in syngeneic C3H female mice.Mice were observed every day for appearance of tumors by scoring thetime of tumor appearance and the size of tumor. After 50 days, mice werekilled and blood was collected by heart puncture. Tumors were excised,weighed and either fixed for pathological examination or quick frozen inliquid nitrogen. Other organs of the mice were also collected andexamined.

Tumorigenicity for antisense clones and control clones was examined andcompared to the wild type PC cells. Table 1 above, shows the resultsobtained with one of them. FIG. 3 shows the picture of tumor bearingmouse detected with 10⁶ control transfected cells and of mouse injectedwith antisense clone. Empty vector control transfected clones maintainedsimilar tumorigenic properties as the parent PC cells, whereas no tumorformation was observed in the mice injected with antisense clones. Evenafter 90 days, these mice still did not present tumors. This experimentwas repeated twice. Moreover, additional clones (2 antisense and 2control) were also examined. The same results were obtained with otherantisense clones.

Antisense cDNA for Human GP88

The approach for stable transfection of antisense GP88 cDNA in PC cellsdescribed above has also been applied to inhibiting GP88 expression inhuman breast carcinoma cell lines.

In this case, the human antisense cDNA construct consisted of a 400 bpcDNA fragment inserted in the antisense orientation in the commerciallyavailable pcDNA3 mammalian expression vector (In Vitrogen, San Diego,Calif.) which contains pCMV4 cytomagelovirus CMV promoter and neomycinresistant gene so that double transfection of pCMV4 and pRSVneo is notrequired like the ones described above for PC cells.

To generate the antisense cDNA fragment the following pairs of primerswith appropriate restriction enzymes sites were synthesized and used inthe PCR reaction:

A-hGP88: (SEQ ID NO: 12) 5′-A10GGATCCACGGAGTTGTTACCTGATC-3′ (positionnt: 362-344) H-hGP88: (SEQ ID NO: 13) 5′-A10GAATTCGCAGGCAGACCATGTGGAC-3′(position nt: −12 to +8)

The amplified cDNA fragment with EcoRI and BamHI restriction sites wasinserted in the antisense orientation in the EcoRI and BamHI sites ofthe pcDNA3 mammalian expression vector. This expression vector constructcalled pCAS was transfected in the human mammary carcinoma cell lineMCF7 and MDA-MB-468 DME-F12 medium supplemented with fetal bovine serumby the calcium phosphate method (55). Selection of transfected cells wasdone by cultivating the cells in the presence of 800 μg/ml of Geneticinto select cells that are neomicin resistant. Neomicin resistant cloneswere picked with cloning rings and passaged in medium supplemented with10% fetal bovine serum (FBS) and with geneticin (800 μg/ml). Transfectedclones selected for their resistance to geneticin were further examinedby methods similar to the ones described above for PC cells transfectedwith mouse GP88 antisense cDNA. The presence of the antisense cDNAconstruct in genomic DNA was checked by PCR analysis using as primersfor the PCR reaction T7 primer in the pcDNA3 expression vector andH-hGP88 primer described above. PCR reaction amplified a 420 bp DNAfragment in cells that expressed the transfected human GP88 antisenseDNA fragment.

Expression of endogenous GP88 was determined by Western blot analysis ofcell lysates and conditioned medium of transfected clones using antiE19V antiserum to select antisense clones with maximum inhibition ofGP88 expression. Selected antisense clones were further analyzed toexamine their growth properties in vitro and in vivo. Transfection ofempty pcDNA3 vector in MCF7 and MDA-MB-468 cells was performed ascontrol for these experiments.

An alternative method to transfection of antisense cDNA is to useantisense oligonucleotides. It is known in the art that sequences aroundthe translation initiation site (ATG encoding the first methionine)provide good sequences for efficient antisense activity. Secondly,sequences with an adequate GC content and that start with either a G ora C have increased efficiency and stability in forming a hybrid withcorresponding sense sequence (32, 37, 38). Based on this rationale, itis anticipated that the following two sequences will be efficacious asantisense oligonucleotides to human GP88. The first one is a 22-mernamed HGPAS1 starting ii nucleotides upstream of the first ATG(methionine codon): HGPAS1 (SEQ ID NO: 14) (22):5′-GGGTCCACATGGTCTGCCTGC-3′. The second oligomer is a 24 mer namedHGPAS2 (24) located 21 nucleotides 3′(downstream) of the first ATG:HGPAS2 (SEQ ID NO: 15) (24):5′-GCCACCAGCCCTGCTGTTAAGGCC-3′. Otheroligonucleotide antisense sequences can be explored by those of ordinaryskill given the teachings herein. To judge the efficacy of a sequence toinhibit GP88 expression, oligonucleotides will be added to breastcarcinoma cells in culture or any other human cell types under study inincreasing doses. Cells will be collected at various time points (12hours to several days) to measure the level of expression of GP88protein by Western blot analysis or EJA using an antihuman GP88antibody, using techniques known to those of ordinary skill in the art.Control cells will be treated with a nonsense or a sense oligomer.

EXAMPLE 10 Inhibition of Tumor Growth in Humans

The present example provides the following:

Comparison of the expression of GP88 mRNA and protein in non tumorigenicmammary epithelial cells MCF10A and in malignant MCF7 and in MDA-MB-468cells.

Growth of malignant MCF7 and MDA-MB-468 cells in the presence ofneutralizing anti-human GP88 antibody (anti-human E19V monoclonalantibody) leads to inhibition of proliferation of cells.

Proliferation in vitro and tumorigenicity in vivo of human breastcarcinoma cells are inhibited by inhibiting GP88 expression by antisenseGP88 DNA.

Comparison of GP88 Expression in Non Tumorigenic Human MammaryEpithelial Cells and in Tumorigenic Breast Carcinoma Cell Lines

We investigated the expression of GP88 mRNA and protein in three humanbreast cell lines: The MCF10A cell line, which is a non tumorigenichuman mammary epithelial cell line, and in two human breast carcinomacell lines, MCF7 which is estrogen receptor positive and MDA-MB-468which is estrogen receptor negative.

Expression of GP88 mRNA expression was done by Northern blot analysis oftotal RNA extracted from these cell lines using a radiolabeled humanGP88 cDNA probe. Expression of GP88 protein was measured by Western blotanalysis of immunoprecipitated GP88 using either rabbit anti-human GP88polyclonal antibody or the anti-human E19V monoclonal antibody. Thislatter antibody was developed by immunizing mice with human peptide E19Vconjugated to protein as described in Example 8.

Anti-human GP88 antibodies that we have now at our disposal are:polyclonal antihuman GP88 antibody developed in rabbits using as antigena 37 kDa fragment of human GP88 expressed as a histidine tagged proteinin bacteria, and the anti-human GP88 antibody (polyclonal andmonoclonal) developed by using as antigen E19V peptide conjugated tokeyhole limpet hemocyanin. Development of these antibodies (polyclonaland monoclonal) are described above. All of these antibodies can be usedfor immunoprecipitation and Western blot analysis of human GP88 in humantissues and cells.

The Northern blot analysis shows that the expression of GP88 mRNA in thenon tumorigenic MCF10A cells is very low and increases at least 5-10times in the human breast carcinoma cell lines MCF7 and MDA-MB-468 (FIG.14).

The results of the Western blot analysis of the three cell lines showthat GP88 protein expression is undetectable in culture medium (CM)collected from the non-tumorigenic MCF10A cells, whereas it increased10-20 times in media conditioned by the MCF7 cells and by the MDA-MB-468cells. In addition to being secreted in the culture medium of the breastcarcinoma cells, GP88 protein is also expressed at high levels in celllysates of the maligant cells, whereas it is undetected in the nontumorigenic MCF10A cells.

These data confirm that human breast carcinoma cells overexpress GP88when compared to non tumorigenic human mammary cells.

Inhibition of Growth of Malignant MCP7 and MDA-MB-468 Cells in thePresence of Neutralizing Anti-human GP88 Antibody (Anti-human E19VMonoclonal Antibody)

Experiments were carried out in which human breast carcinoma MCF7 cellswere incubated with different doses of anti-human GP88 monoclonalantibody (anti-E19V peptide IgG fraction). Proliferation was measured by[³H] thymidine incorporation into DNA. Control cells consisted of cellsincubated with the same amount of unrelated mouse IgG. Results show thatincubation of MCF7 cells with 25 μg/ml of anti-GP88 monoclonal antibodyleads to a 50% inhibition of thymidine incorporation, whereas 100 μg/mlresulted in an 80% inhibition of proliferation. Similar results werefound as indicated above for MDA-MB-468 cells. These data again confirmthat (i) anti-human E19V antibody neutralizes human GP88, and (ii)malignant human cells which secrete high levels of GP88 in culturemedium and which require GP88 to proliferate can be effectivelyinhibited by treatment with anti-human GP88 neutralizing antibody, thusdemonstrating that inhibition of GP88 action on tumor cells is aneffective therapy for inhibiting tumor growth of cells overexpressingGP88.

Proliferation In Vitro and Tumorigenicity In Vivo of Human BreastCarcinoma Cells are Inhibited by Inhibiting GP88 Expression by AntisenseGP88 DNA

Human breast carcinoma MDA-MB-468 cells were stably transfected withantisense human GP88 cDNA construct (400 bp fragment inserted intopcDNA3 expression vector). This construct is described in Example 9.Stable antisense clones were isolated and characterized. In particular,antisense clones were selected based on the fact that expression of GP88was effectively inhibited. This was determined by measuring GP88expression in antisense cells and empty vector control cells by Westernblot analysis of cell extracts and conditioned medium using antiGP88antibody. Several clones were obtained and characterized. Similarresults were obtained with all antisense clones isolated. Data presentedhere concern one antisense clone called 468AS. As shown in FIG. 15,transfection of antisense GP88 cDNA in MDA-MB-468 cells (468AS) resultedin inhibition of GP88 protein expression when compared to empty vectorcontrol MDA-MB-468 cells (468 Cont).

Measurement of the proliferation rate of antisense GP88 and empty vectorcontrol cells indicated that antisense cells had an 80% inhibition ofcell proliferation when compared to the empty vector transfected cells.Our data with all the clones analyzed also showed that the extent ofgrowth inhibition was directly correlated to the degree of inhibition ofexpression of GP88 in the antisense clones.

Antisense human breast 468AS cells which displayed the highestinhibition of GP88 expression, and empty vector control (468 Cont) cellswere injected subcutaneously into female nude mice in the breast area ata density of 2×10³ cells/mouse using 4 mice per cell line. Tumorappearance was monitored by visually inspecting the mice. After 4 weeks,the mice were sacrificed, tumors were excised and weighed (see Table 3below).

TABLE 3 Effect of inhibition of GP88 expression by antisense GP88cDNAtransfection on tumor growth of human breast cancer cells MDA-MB-468 innude mice Cells Mice bearing tumors Weight of tumors (g) 468 Cont empty4/4  0.2 ± 0.06 vector 468AS antisense GP88 3/4 0.05 ± 0/025** (**p ≧0.01 significant)The results show that inhibition of GP88 expression resulted in a 75%inhibition of tumor growth for human breast carcinoma cells.

This experiment demonstrated that inhibition of GP88 expression in humanbreast carcinoma cell lines leads to inhibition of tumorigenicity.

EXAMPLE 11 Diagnostic Test for Tumorigenicity

In teratoma and in breast cancer, an increase in tumorigenic propertiesis associated with an increase in GP88 expression or an increase in GP88responsiveness.

Moreover, FIG. 4 shows that the level of expression of GP88 in tumortissue is increased when compared to the surrounding tissues.Accordingly, increase of GP88 level can be used as a diagnostic approachto detecting tumor. In human tumor biopsies, a change (increase) in GP88expression when compared to the level of GP88 in normal correspondingtissues is indicative of the state of tumorigenicity or malignancy ofthe tissue biopsy analyzed. Increase in expression of GP88 can bemeasured at the mRNA level or at the protein level. GP88 mRNA expressioncan be measured either by Northern blot analysis, RNAse protection assayor RT-PCR

GP88 protein expression is quantitated by ELISA, EIA or RIA using ananti-GP88 antibody.

The ability to measure GP88 expression in tissue extracts in comparisonto corresponding tissues from normal subject can be used to predict thedegree of tumorigenicity of a particular cancer or to determine whetherthis particular cancer will be responsive to anti-GP88 therapy.

For diagnosis of diseases associated with increase in GP88responsiveness, tissue biopsies to be analyzed will be treated withanti-GP88 neutralizing antibodies or anti-GP88 receptor antibodies tosee if such treatment inhibits growth of the cells. Alternatively, theability of GP88 antisense oligonucleotides to inhibit growth indicatesthat expression of GP88 is required for growth in vivo.

EXAMPLE 12 Characteristics of GP88 Cell Surface Receptors

Binding of iodinated GP88 to a variety of cell lines CCL64, 1246, PC andthe mammary epithelial C57MG was determined in order to examine thebiochemical characteristics of GP88 cell surface receptors using themethods described below. For these studies we used affinity purifiedrecombinant GP88 (rGP88) from the culture medium of baculovirus infectedSP9 insect cells as ligand. Methods to prepare rGP88 have previouslybeen described.

a) Iodination of GP88.

Recombinant GP88 (rGP88) was iodinated by the chloramine T method at 4°C. Other known methods can also be applied. Briefly, 1 μg of GP88 wasincubated for 2 min with ¹²⁵I Na (100 μCi) that had been preincubatedfor 90 seconds with 2 μg chloramine T. The reaction was quenched withthe addition of 100 μl saturated tyrosine, 10 μl of a 1% solution ofbovine serum albumin (BSA) and 2 μg of sodium metabisulfite. Afteraddition of 100 μl PBS, the iodinated protein was separated from free Na¹²⁵I by gel filtration on a Sephadex-G50 column that had beenpreincubated with PBS containing 1% bovine serum albumin and thenextensively washed with PBS to reduce non-specific binding. The labeledproteins were eluted with PBS and fractions monitored for radioactivity.Amount of incorporated radioactivity was estimated by TCA precipitation.Specific activity of ¹²⁵I-GP88 was typically 30-50 μCi/μg. This methodand other methods for iodination of proteins are well known to peopleskilled in the art can also be used to iodinate PC derived GP88 ratherthan rGP88 or any derivatives thereof.

b) ¹²⁵I-GP88 Binding.

The example provided here describes binding assay with the mink lungepithelial cell line CCL64 but has also been applied to several celllines including the 1246, PC cell lines and the mammary epithelial cellline C57MG. The binding assays were performed on cell suspension. CCL64cells were cultivated as monolayer in DME medium supplemented with 10%fetal bovine serum (FBS) until they reached confluency. At that time,cells were washed with PBS and detached by incubation with a solution of0.25 mg/ml of trypsin and 1 mM EDTA. The cells were harvested bycentrifugation, washed with culture medium and counted with ahemocytometer. For binding assays, 10⁶ cells were resuspended in 500 μlof binding buffer consisting of DME medium supplemented with 1% bovineserum albumin in 1.5 ml ependorf tubes and incubated for 2 hours at 25°C. with 10⁵ cpm of ¹²⁵I-rGP88 and increasing concentrations of coldrGP88 from 1 to 100 ng/ml. Binding was stopped by centrifuging the cellsfollowed by 3 successive washings at 4° C. with cold binding bufferfollowed by centrifugation. Cell pellets were counted with a gammacounter. Scatchard analysis of binding data was carried out by computerLigand program. Values correspond to the average of three separateexperiments with duplicate determinations per experimental point.Scatchard analysis of binding of ¹²⁵I-GP88 to CCL64 cells wascurvilinear corresponding to the presence of two classes of cell surfacereceptors: a high affinity class with a Kd of 4.3±1.5×10⁻¹¹ M, 560±170sites/cell and a low affinity class of receptors with a Kd of3.9±1.9×10⁻⁹M, 17,000±5900 sites/cell.

c) Cross-Linking Studies of ¹²⁵I-GP88 to CCL64 and Other Cell Lines.

Cross-linking of ¹²⁵I-GP88 to cell surface receptors was carried outusing disuccinimidyl suberate (DSS). For cross-linking studies, 5×10⁵cells were suspended in 250 μl of binding buffer in eppendorf tubes.¹²⁵I-GP88 was added in 50 μl of binding buffer (DME medium with 1% BSA)with or without 100 fold excess unlabeled GP88 ligand. Binding wasperformed as described in the previous paragraph. At the end of theincubation period, the cells were washed one time with 0.2% BSA-DME andone time with PBS before cross-linking was carried out. Dissucinimidyesuberate (DSS) was dissolved in DMSO at a 100 mM just prior to use. Thecells were resuspended in 200 μl PBS containing 1 mM disuccinimidylsuberate (DSS,) at room temperature for 15 min. After crosslinking, thecells were centrifuged, washed and extracted with 25 μl extractionbuffer (PBS containing 1% Triton X-100, 0.1% SDS, 0.5 mMphenylmethylsulfonyl fluoride (PMSF) at 4° C. Samples were centrifugedfor 5 min at 13,000×g and 25 μl of supernatant from each sample wasmixed with 4 μl of 20% SDS and 15 μl 3× Laemmli's sample buffer (56)containing b-mercaptoethanol and boiled for 5 min. Electrophoresis ofsamples was carried out on 7% SDS polyacrylamide slab gel according toLaemmli (56) using a minigel apparatus (Bio-Rad, Richmond, Calif.). Thedried gels were exposed to X-ray films for autoradiography at −70° C.

As shown in FIG. 12, autoradiographic analysis revealed the presence ofone major cross-linked band with a molecular weight of 190-195 kDa. Thiswould correspond to a molecular weight for the unbound receptor of about110 kDa for the major band. The intensity of the major cross-linked bandwas decreased in the lanes where binding was carried out in the presenceof excess cold GP88. Cross-linked band could not be detected ifexperiment and gel electrophoresis was performed in the absence of DSS.Additional experiments using samples treated or not withb-mercaptoethanol prior to performing the electrophoresis indicated thatthe cell surface receptors for epithelin/granulin precursor aremonomeric.

Cross-linking of ¹²⁵I-GP88 to cell surface receptors were also carriedout with 3T3, PC cells and the mammary epithelial cells C57MG by thesame method. The results of these experiments indicated similarcross-linking pattern for GP88 in all cell lines tested suggesting thepresence of cell surface binding sites with similar size in fibroblasticand epithelial cells (FIG. 13)

GP88 Mediated Signal Transduction in Mammary Epithelial Cell Line C57MG

Experiments to determine the characteristics of the signal transductionpathway activated by GP88 after binding to its cell surface receptorswere carried out with GP88 in the mammary epithelial cell line C57MG. Wehave shown that anti-K19 T antibody can immunoprecipitate the complexformed by GP88 and its cell surface receptors on various cell types andin the mammary epithelial cells C57MG in particular. This feature hasallowed us to further characterize biochemical properties of GP88receptor and the signal transduction it mediates leading to growthstimulation. We have shown that GP88 receptors belong to the tyrosinekinase family of receptors. Upon binding of GP88 to its cell surface,GP88 receptor is activated by phosphorylation on tyrosine residuesresulting in phosphorylation of several signaling molecules includingIRS-1, SHC, Grb2 and leading to activation of MAP kinase ERK-2.

Having now fully described this invention, it will be appreciated bythose skilled in the art the same can be performed within a wide rangeof equivalent parameters concentrations and conditions without departingfrom the spirit and scope of the invention and without undueexperimentation.

REFERENCES

-   1. Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K,    Watson, J. D., 1983 Molecular Biology of the Cell. Garland    Publishing Inc. NY.-   2. Cross, M and Dexter, T (1991) Growth Factors in development,    transformation and tumorigenesis. Cell, 64, 271-280.-   3. Sporn, M. B. and Todaro, G. J. (1980) Autocrine secretion and    malignant transformation. New. Engl. J. Med. 303, 878-880.-   4. Zhou, J., Gao, G., Crabb, J. W. and Serrero, G. (1993)    Purification of an autocrine growth factor homologous with mouse    epithelin precursor from a highly tumorigenic cell line. J. Biol.    Chem. 268, 10863-10869.-   5. Plowman, G. D., Green, J. M. Neubauer, M. G., Buckley, S. D.,    McDonald, V. L., Todaro, G. J., Shoyab, M. (1992). The epithelin    precursor encodes two proteins with opposing activities on    epithelial cell growth. J. Biol. Chem. 267, 13073-13078.-   6. Bateman, A., Belcourt, D., Bennett, H., Lazure, C. and    Solomon, S. (1990) Granulins, a novel class of peptide from    leucocytes. Biochem. Biophys. Res. Commun. 173, 1161-1168.-   7. Nestor, J J Jr. Newman, S R, DeLustro, B., Todaro, G J,    Schreiber, A B, 1985, A synthetic fragment of rat transforming    growth factor alpha with receptor binding and antigenic properties.    Biochem. Biophys. Res. Commun. 129, 226-232.-   8. Adelman, J P, Hayflick, J S, Vasser, M, Seeburg, P H (1983) In    vitro deletional mutagenesis for bacterial production of the 20,000    dalton form of human pituitary growth hormone. DNA, 2, 183-193.-   9. Serrero, G. and J. C. Khoo (1982) An in vitro model to study    adipose differentiation in serum-free medium. Anal. Biochem.    120:351-359.-   10. Serrero, G. (1984). Study of a teratoma-derived adipogenic cell    line 1246 and isolation of an insulin independent variant in    serum-free medium. In: Hormonally Defined Media, a Tool in Cell    Biology, (G. Fisher and R J. Wieser, eds.), Springer-Verlag, New    York, Berlin, Heidelberg, Tokyo, pp. 310-313.-   11. Serrero, G. (1985) Tumorigenicity associated with loss of    response to insulin and of differentiation ability in the adipogenic    cell line 1246. In Vitro Cell. Devel. Biol. 21:537-540.-   12. Serrero, G., Zhou, J., Mills, D. and Lepak, N. (1991) Decreased    transforming growth factor □ response and binding in    insulin-independent, teratoma-derived cell lines with increased    tumorigenic properties. J. Cell Physiol. 149 503-511.-   13. Arteaga, C. L. (1992) Interference with the IGF system as a    strategy to inhibit breast cancer growth. Breast Cancer Res. Treat    22, 101-106.-   14. Arteaga, C. L., and Osborne, C. k. (1989) Growth inhibition of    human breast cancer cells in vitro with an antibody against the type    1 somatomedin receptor. Cancer Res. 49, 6237-6241.-   15. Schofield, P N, Granerus, M, Tally, M, Engstrom, W (1994) The    biological effects of a high molecular weight form of IGF-II in a    pluripotential human teratocarcinoma cell line. Anticancer Res. 14,    533-538.-   16. Trojan, J, Johnson, T R, Rudin, S D, Blossey, B K, Kelley, K M,    Shevelev, A, Abdul-Karim, F W, Anthony, D D, Tykocinski, M L, Ilan,    J et al (1994) Gene therapy of murine teratocarcinoma: separate    functions for insulin-like growth factors I and II in immunogenicity    and differentiation. Proc. Natl. Acad. Sci. USA 91, 6088-6092.-   17. Trojan, J, Johnson, T R, Rudin, SD, Ilan, J, Tykocinski, M L,    Ilan, J (1993) Treatment of rat glioblastomas by immunogenic C6    cells expressing antisense insulin-like growth factor I RNA.    Science, 259, 94-97.-   18. Kohler, G and Milstein, C. 1975: Continuous culture of fused    cells secreting antibody of predefined specificity. Nature 256,    495-497.-   19. de St. Groth, F. and Scheidegger, D. (1980) Production of    monoclonal antibodies. Strategy and tactics. J. Immunol. Methods. 35    1-21.-   20. Harlow, E. and Lane D. (1988) Antibodies. A laboratory manual.    Cold Spring Harbor Laboratory. Cold Spring Harbor, N.Y.-   21. Cadilly, S., Riggs, A. D., Pande, H., Shively, J. E., Holmes, W.    E., Rey. M. Perry, L. J., Wetzel, R, Heyneker, H. L. (1984)    Generation of antibody activity from immunoglobulin polypeptide    chains produced in Escherichia Coli. Proc. Natl. Acad. Sci. USA. 81,    3273-3277.-   22. Morrison SL, Johnson, M J., Herzenberg, L A, Oi, V T (1984)    Chimeric human antibody molecules: mouse antigen-binding domains    with human constant region domains. Proc. Natl. Acad. Sci USA. 81,    6851-6855.-   23. Liu, A Y, Robinson, R R, Hellstrom, K E., Murray, E D Jr.,    Chang, C P, Hellstrom, T. (1987) Chimeric mouse-human IgG antibody    that can mediate lysis of cancer cells. Proc. Natl. Acad. Sci. USA    84, 3439-3443.-   24. Better, M, Chang, C P, Robinson, R R Horwitz, A H. 1988.    Escherichia Coli secretion of an active antibody fragment. Science,    240, 1041-1043.-   25. Riechmann, L., Clark, M., Waldmann, H. and Winter, G. (1988)    Reshaping human antibodies for therapy. Nature, 332, 323-327.-   26. Baca, M., Presta, L. G., O'Connor, S. J., Wells, J. A. (1997)    Antibody humanization using monovalent phage display. J. Biol. Chem.    272, 10678-10684.-   27. Rosok, M. J., Yelton, D. E., Harris, L. J., Bajorath, J.,    Hellstrom, K-E., Hellstrom, I., Cruz, G. A., Kristensson, K, Lin,    H., Huse, W. D., and Glaser, S. M. (1966) A combinatorial library    strategy for the rapid humanization of anticarcinoma BR96 Fab. J.    Biol. Chem. 271, 22611-22618.-   28. Wahl, R. L., Parker, C. W., Philpott, G. W., J. Nucl. Med. 1983,    24, 316-325 Improved radioimaging and tumor locations with    monoclonal F(ab′)2.-   29. Mulshine, J. L., Avis, I., Treston, A. M., Mobley, C.,    Kaspryzyk, P., Carrasquillo, J. A., Larson, S. M., Nakanishi, Y.,    Merchant, B., Minna, J. D., et al (1988) Clinical use of a    monoclonal antibody to bombesin-like peptide in patients with lung    cancer. Ann. NY Acad. Sci. 547, 360-372.-   30. Munroe, S. H., 1988, EMBO. J. 7:2523-2532 Antisense RNA inhibits    splicing of prem-RNA in vitro.-   31. Mullis, K B, Faloona, F A (1987) Specific synthesis of DNA in    vitro via polymerase catalyzed chain reaction. Met. Enzymol. 155,    335-350.-   32. Mercola, D., and Cohen, J. S. (1995) Antisense approaches to    cancer gene therapy. Cancer Gene Therapy 2 47-59.-   33. Wagner, R W. (1994) Nature, 372, 333-335 Gene Inhibition using    antisense aligodeoxy-nucleotides.-   34. Wagner, R. W. (1995) The state of the art in antisense research.    Nature Medicine 1, 1116-1118-   35. Sambrook et al. Molecular Cloning. A Laboratory Manual. Cold    Spring Harbour, N. Y. (1989)-   36. Brysch, W. and Schlingensiepen, K. H. (1994) Design and    application of antisense oligonucleotides in cell culture, in vivo    and as therapeutic agents. Cell Mol. Neurobiol. 14, 557-568.-   37. Helene, C (1991) Rational design of sequence-specific oncogene    inhibitors based on antisense and antigene nucleotides. Eur. J.    Cancer 27, 1466-1471.-   38. Giles, R V., Spiller, D. G., Green, J. A., Clark, R E. and    Tidd, D. M. (1995) Optimization of antisense oligodeoxynucleotide    structure for targeting bcr-abl mRNA. Blood 86, 744-754.-   39. Thaler, D. S., Liu, S. and Tombline, G. (1996) Extending the    chemistry that supports genetic information transfer in vivo:    phophorothioate DNA, phosphorothioate RNA, 2-O-methyl RNA and    methylphosphonate DNA. Proc. Natl. Acad. Sci. USA. 93, 1352-1356.-   40. Gryaznov, S., Skorski, T., Cucco, C., Nieborovska-Skorska, M.,    Chiu, C. Y., Lloyd, D., Chen, J. K., Koziolkiewicz, M. and    Calbretta, B. (1996) Oligonucleotide N3′-->P5′ phosphoramidates as    antisense agents. Nucleic Acid Res. 24, 1508-1514.-   41. Lappalainen, K., Urtti, A., Soderling, E., Jaaskelainen, I.,    Syrjanen, K. and Syrjanen, S. (1994) Cationic liposomes improve the    stability and intracellular delivery of antisense oligonucleotides    into CaSki cells. Biochim. Biophys. Acta. 1196, 201-208.-   42. Ensoli, B. et al. (1994) Block of AIDS-Kaposi's sarcoma KS cell    growth, angiogenesis and lesion formation in nude mice by antisense    oligonucleotide targeting basic fibroblast growth factor. A novel    strategy for the therapy of KS. J. Clin. Inves. 94, 1736-1746.-   43. Peng, B., Methta, N H., Fernandes, H., Chou, C C. and    Raveche, E. (1995) Growth inhibition of malignant CD5+B (b-1) cells    by antisense IL-10 oligonucleotide. Leukemia Res. 19 159-167.-   44. Glick, B. R (1987) J. Ind. Microbiol, 1:277-282.-   45. Cenatiempo, Y., (1986) Biochirnie, 68: 595-516 Prokaryotic gene    expression in vitro: transcription-translation coupled systems.-   46. Gottesman, S (1984) Ann. Rev. Genet. 18:415-442 Bacterial    regulation: global regulatory networks.-   47. Hamer, D. H. and Walling, M., 1982, J Mol Appli. Gen. 1: 273-288    Regulation in vivo of a closed mammalian gene: cadmium induces the    trancription of a mouse metallothionein gene in SV40 vectors.-   48. McKnight, S., 1982, Cell, 31: 355-365 Functional relationships    between transcriptional control signals of the thymidine kinase gene    of herpes siplex virus.-   49. Johnston, S. A. and Hopper, J. E. 1982 Proc. Natl. Acad. Sci.    USA, 79, 6971-6975 Isolation of the yeast regulatory gene, GALA and    analysis of its dosage effects on the galactose/melibiose regulon.-   50. Benoist, C., and Chambon, P., 1981 Nature, 290: 304-310 In vivo    sequence requirements of the SV40 early promoter region.-   51. Andersson, S., Davis, D. L., Dahlback, H., Jornvalif, H. and    Russell, D. W. 91989) Cloning, Structure and Expression of the    Microchondrial cytochrome P450 sterl 26 hydroxylase a bile acid    biosynthetic enzyme, J. Biol. Chem. 264, 8222-8229.-   52. Chomczinski, P. Sacchi, N (1987) Single-step method of RNA    isolation by acid guanidinium thiocyanate-phenol-chloroform    extraction. Anal. Biochem. 162, 156-159. 53. Sepp-Lorenzino, L.,    Rosen, N., Lebwohl, D. E. (1994) Insulin and insulin-like growth    factor signaling are defective in the MDA-MB-468 human breast cancer    cell line. Cell Growth Diff 5, 1077-1083.-   54. Culouscou, J-M., Carlton, G. W., and Shoyab, M. (1993)    Biochemical analysis of the epithelin receptor. J. Biol. Chem. 268,    10458-10462.-   55. Ausubel, F. M., Brent, R E., Moore, D. D., Smith, J. A.,    Seidman, J. G., and Struhl, K. (1987). Current Protocols in    Molecular Biology, Greene Publishing and Wiley Interscience, New    York.-   56. Laemmli. UK (1970) Cleavage of structural proteins during    assembly of the head of bacteriophage T4. Nature, 227, 680-685. 57.    Siegall, C. B. 1994 Targeted Toxins as Anticancer Agents, Cancer, 74    1006-1012.

1. A method of inhibiting the growth of a tumor cell in a mammal comprising administering a PC Cell Derived Growth Factor antisense oligonucleotide to the tumor cell by injection of said oligonucleotide to said mammal, wherein said oligonucleotide inhibits the growth of the tumor cell, and wherein said oligonucleotide is an oligonucleotide targeted to at least a portion of SEQ ID NO: 16 around the translation initiation site.
 2. The method of claim 1, wherein the tumor cell is a breast carcinoma cell.
 3. The method of claim 1, wherein said oligonucleotide is about 15-30 nucleotides in length.
 4. A method of decreasing the size of a tumor comprising administering a PC Cell Derived Growth Factor antisense oligonucleotide to the tumor wherein said oligonucleotide decreases the size of the tumor, and wherein said oligonucleotide is an oligonucleotide targeted to at least a portion of SEQ ID NO: 16 around the translation initiation site.
 5. The method of claim 4, wherein said tumor is a breast tumor.
 6. The method of claim 4, wherein said oligonucleotide is about 15-30 nucleotides in length.
 7. A method of inhibiting the expression of PC Cell Derived Growth Factor protein in a cell comprising administering a PC Cell Derived Growth Factor antisense oligonucleotide to the cell wherein said oligonucleotide inhibits the expression of PC Cell Derived Growth Factor protein, and wherein said oligonucleotide is an oligonucleotide targeted to at least a portion of SEQ ID NO: 16 around the translation initiation site.
 8. A method of inhibiting the proliferation of a tumor cell comprising administering a PC Cell Derived Growth Factor antisense oligonucleotide to the cell wherein said oligonucleotide inhibits the proliferation of the tumor cell, and wherein said oligonucleotide is an oligonucleotide targeted to at least a portion of SEQ ID NO: 16 around the translation initiation site.
 9. The method of claim 8, wherein the proliferation of the tumor cell is inhibited by about 80 percent.
 10. The method of claim 8, wherein said tumor cell is a breast carcinoma cell.
 11. A method of inhibiting the expression of PC Cell Derived Growth Factor protein in a cell comprising administering a PC Cell Derived Growth Factor antisense oligonucleotide comprising SEQ ID NO: 14 wherein said oligonucleotide inhibits the expression of PC Cell Derived Growth Factor protein. 